bluuebunny/crossref_metadata_2025 huggingface dataset:
Intestinal Inflammation and Seizure: understanding the role of TNF‐alpha in rat model
Authors: Prakash Ajay , MEDHI BIKASH , Avti Pramod Kumar , CHAKRABARTY AMITAVA , Khanduja Kishan Lal
Published: June 2021

Objective: Present study was carried out to evaluate the role of TNF α and correlation between intestinal inflammation and PTZ induced seizure susceptibility in rat model.

Methods and Materials: Rats were divided into 3 groups (n=6 in each group). Colitis was induced by single intra‐colonic administration of TNBS (20mg / 35% ethanol). Group I received ethanol (vehicle) intra colonic, Group II (TNBS), Group III (thalidomide 150mg/kg b. wt/d orally for 14 days after induction of colitis). After 14 days animals were sacrificed and colonic tissue was used for morphological score, histological score and different biochemical parameters (MPO, MDA, SOD, Catalase, GPx, TNF‐α in serum & brain).

Results: Seizure susceptibility showed increase seizure score as well as reduction of onset of the seizure in the colitis group compared to control group. Thalidomide provided protection against TNBS‐induced colonic damage both histologically, morphologically and as evident in different biochemical parameters (MPO, MDA SOD, CAT, GPx, and TNF‐α in serum and brain) (p<0.001).

Conclusion: Present study showed the increase seizure susceptibility following peripheral inflammation of colitis indicate correlations of colitis and seizure with role of TNF α. (Supported by PGIMER, Chandigarh, India)
Role of NOS‐NO signaling in clonidine‐ethanol evoked synergistic behavioral impairment
Authors: Bender Tara Summer , Abdel‐Rahman Abdel A
Published: June 2021

Clonidine‐ethanol combination induces synergistic behavioral impairment in rats as measured by rotorod performance and loss of righting reflex (LORR) duration. We tested the hypothesis that NOS‐NO signaling in the locus ceruleus (LC) is implicated in this interaction. Pretreatment (i.c.) with non‐selective NOS inhibitor (L‐NAME) or nNOS‐selective inhibitor (NPLA) enhanced the impairment of rotorod performance caused by clonidine‐ethanol combination. Prolongation of behavioral impairment was caused by L‐NAME enhancement of the effect of ethanol, not clonidine. LORR caused by clonidine‐ethanol combination was abolished by eNOS‐selective inhibition (L‐NIO), but not nNOS inhibition. Western blot analyses complemented the pharmacological evidence by demonstrating that clonidine‐ethanol combination inhibits phosphorylation (activation) of nNOS (p‐nNOS) and increases levels of phosphorylated eNOS (p‐eNOS) in the LC; the change in p‐nNOS, but not p‐eNOS, was paralleled by similar change in LC p‐ERK1/2. Therefore, NOS‐derived NO in the LC is differentially involved in underlying mechanisms for clonidine‐ethanol induced behavioral impairment. A decrease in nNOS‐derived NO contributes to impaired rotorod performance, while an increase in eNOS‐derived NO contributes to LORR duration elicited by clonidine‐ethanol combination. [Supported by NIH grants 5F31AA015472 (TSB) and 2R01AA07839 (ARA)]
A GABA‐A alpha1 third transmembrane cysteine mutant with unusual sensitivity to allopregnanolone
Authors: Williams Daniel B
Published: June 2021

GABA‐A receptors are known to be modulated by neurosteroids. At wild type receptors, low concentrations of allopregnanolone can potentiate GABA currents while higher concentrations can activate GABA‐A receptors. Regions of the receptor involved in the binding or transduction of steroid are unclear. Regions may be N terminal to M2 or in M4 or in the cavities between transmembrane domains. While investigating the third transmembrane domain (M3) of the rat alpha1 subunit, I discovered a cysteine mutant that has unusual sensitivity to allopregnanolone. When expressed with wild type beta1 and gamma2s subunits, a mutant in alpha1 M3, S299C, showed activation by allopregnanolone with an EC50 in the range of 60–90 nM. This EC50 is normally in the range for potentiation at wild type receptors. This data could allow a parallel to the data gathered for ethanol at S270 and A291: to pinpoint a potential binding site or transduction area for neurosteroids. Further, this residue would likely be located in the cavities between transmembrane domains where some steroid actions have been localized.
Maturation and Pharmacological Properties of Postsynaptic GABAA Receptors
Authors: Wang Haiying , Griffith William H. , McDermott Angelika N. , Murchison David , Frye Gerald D.
Published: June 2021

Medial Septum/Diagonal Band (MS/DB) neurons display age‐dependent acceleration of GABA miniature postsynaptic current (mPSC) frequency, amplitude and decay rate in brain slices over postnatal day (PD) 4–16 (DuBois and Frye SfN Abst, 34: 913.11, 2004). ‘Binge‐like’ alcohol exposure on PD 4–9 equivalent to 3rd trimester of human pregnancy blunted this maturation, possibly by disrupting clustering of α1 containing GABAARs at synapses (Wang and Frye SfN Abst, 34: 570, 2006). In the present study, potentiation by zopidem (0.3–100 μM), a positive allosteric modulator with preferred selectivity for α1 subunit containing GABAARs, was examined in MS/DB neurons in brain slices from PD 4–9 Naïve pup and older rats of 3–4, 17 or 24 months. Zolpidem potentiation after binge‐like ethanol on PD 4–9 (5.25g/kg/day) was investigated during PD 11–16 and PD 80–85. GABAAR–mediated mPSCs were recorded in presence of zolpidem and amplitude, frequency and decay kinetics were analyzed. Zolpidem (0.3–100 μM) showed an age‐dependent potentiation of mPSC decay during PD 4–16 and plateaued in adulthood. The fast decay potentiation by zolpidem in adult and old rats resembled that of PD 4–9 naïve pups; Potentiation of the slow decay resembled that of PD 11–16 control rats. Binge ethanol exposure on PD 4–9 blunted zolpidem potentiation on PD 11–16, which persists through PD 80–85. No gender differences in baseline or zolpidem sensitivity on PD 11–16 were observed after binge ethanol treatment on PD 4–9. These results suggest binge ethanol exposure in developing MS/DB neurons appears to cause long‐term distortion of pharmacological sensitivity of postsynaptic GABAARs, which could contribute to cognitive deficits identified in fetal alcohol spectrum disorders. Supported in part by NIH R56AA12386 (GDF), RO1 AG007805(WHG), T32 MH65728 (ANM)
20‐HETE agonist, N‐[20‐hydroxyeicosa‐5(Z),14(Z)‐dienoyl]glycine, opposes the endotoxin‐induced fall in blood pressure and the decrease in vascular reactivity and lipid peroxidation and increased catalase activity via inhibition of NO production in rats
Authors: Tunctan Bahar , Korkmaz Belma , Buharalioglu C Kemal , Sahan‐Firat Seyhan , Anjaiah Siddam , Falck John R , Yildirim Hatice , Tamer Lulufer , Roman Richard J , Malik Kafait U
Published: June 2021

Endotoxemic shock is a systemic inflammatory response associated with increased NO production and decreased in the expression and activity of CYP4A protein involved in 20‐HETE production, which may contribute to a fall in blood pressure (BP) and vascular reactivity (VR). NO is also known to have both stimulatory and inhibitory effects on lipid peroxidation and activities of antioxidant enzymes. We investigated whether a stable analogue of 20‐ETE, N‐20‐ydroxyeicosa‐(Z),14(Z)‐ienoyl]glycine (5,14‐EDGE), prevents the effects of NO on the fall in BP and VR as well as the changes in lipid peroxidation and catalase activity in rats treated with endotoxin (ET). ET‐nduced increase in nitrite levels and catalase activity in serum, kidney, heart, thoracic aorta (TA) or superior mesenteric artery (SMA) were associated with a decrease in BP, norepinephrin‐enduced contractions in TA and SMA, and malondialdehyde levels in the tissues. These effects of ET were prevented by 5,14‐EDGE. An antagonist of 20‐ETE, 20‐hydroxyeicosa(Z),15(Z)‐dienoic acid, reversed the above effects of 5,14‐EDGE. These data suggest that a decrease in 20‐ETE production may contribute to the effects of NO to reduce BP and VR associated with decreased lipid peroxidation and increased catalase activity during endotoxemia. This study was supported by Mersin University, NIH and the Robert A. Welch Foundation.
Stimulation of endothelial ecto 5′‐nucleotidase by lipopolysaccharides via phosphatidylinositol‐3 pathway
Authors: Leung George P.H. , Man Ricky Y.K. , Vanhoutte Paul , Li Rachel W.S.
Published: June 2021

Inflammation of endothelium is associated with atherosclerosis. It is known that adenosine possesses anti‐inflammatory function and its level is increased at the sites of inflammation. The mechanism underlying the increased adenosine level is not fully understood. Therefore, we studied the effect of inflammation on endothelial ecto‐5’nucleotidase (ecto‐5’Nu), an enzyme which metabolizes extracellular adenosine monophosphate into adenosine. Lipopolysaccharide (LPS) was used to induce inflammation of human umbilical vein endothelial cells (HUVEC). Biochemical assay revealed that the ecto‐5’Nu activity in HUVEC was stimulated by LPS after 24 hr treatment. RT‐PCR showed that mRNA expression of ecto‐5’Nu was not changed but western blot showed that its protein expression was increased by LPS. In addition, LY294‐002, a phosphatidylinositol 3‐kinase (PI3K) inhibitor, reduced the LPS‐stimulated ecto‐5’Nu activity but ammonium pyrrolidine dithiocarbamate (APDC), a NF‐êB inhibitor, had no effect. The stimulation of ecto‐5’Nu by LPS was unaltered when HUVEC was incubated in 25 mM glucose, a condition which mimics the hyperglycemia of diabetes. In conclusion, it is suggested that the increased extracellular adenosine level during inflammation may be related to increased translational regulation of ecto‐5’Nu through a PI3K signaling pathway and this mechanism may not be affected in diabetes.
Ramipril treatment improves vascular endothelial function in db/db mice
Authors: Liang Willmann , Tan Calista Yu Rong , Wright James M. , Laher Ismail
Published: June 2021

Cardiovascular complications are preceded by endothelial dysfunction in type 2 diabetes. This is associated with increased plasma reactive oxygen species (ROS). High ROS levels reduce nitric oxide bioavailability and attenuate vasodilation. The ACE inhibitor ramipril is frequently used in managing the cardiovascular complications of diabetes. We hypothesized that ramipril treatment improves ACh‐induced, endothelium‐dependent vasodilation in db/db mice. We treated 5‐week‐old wild‐type (WT) and type 2 diabetic (db/db) mice with vehicle or ramipril (10mg/kg/day) for 6 weeks. Plasma ROS levels were reduced by ramipril in WT (34%) and db/db (51%) mice. Ramipril treatment increased ACh‐induced vasodilation by 4‐fold in db/db mice without affecting endothelium‐independent vasodilation. These data support a key role for angiotensin II in mediating endothelial dysfunction in diabetes through the generation of ROS. (Supported by Heart and Stroke Foundation of Canada & Singapore Ministry of Education Academic Research Fund)
Modification of the pressor effect of sympathomimetic drugs by carvedilol: the role of nitric oxide and propranolol
Authors: Simaan Joseph A. , Hitti Lourdes
Published: June 2021

The pressor effect of single intravenous doses of norepinephrine (NE 0.05, 0.1, 0.2 ug) and tyramine (T 0.025, 0.5, 0.1 mg) was assessed in anesthetized Sprague‐Dawley rats under pentobarbital anesthesia by measuring systolic carotid pressure before and after carvedilol (C), a beta‐ and alpha1‐adrenoceptor blocker with arteriolar dilating action, given in cumulative doses to produce maximal changes. C (1.36±0.1 mg/kg N=8) reduced the pressor effect of NE and T in all doses by 15–36% and 33–41% respectively that was prevented by subsequent blockade of nitric oxide synthesis with nitro‐L‐arginine (NA 15 mg/kg) and restoration of the basal level of nitric oxide with nitroglycerin (NG) a nitric oxide donor, without further change after propranolol (P 1 mg/kg). The effect of C was also prevented after P (N=9) by 8–27% and 32–42% respectively. NA‐NG was without further effect. C given after P (N=10) potentiated the pressor effect of NE and T by 44–59% and 17–41% respectively without change after NA‐NG. C enhanced the depressor effect of acetylcholine (AC 0.1–0.5 ug) by 15–32% (N=8) which was prevented by NA‐NG. It is concluded that C enhances the synthesis of nitric oxide which decreases the pressor effect of NE and T and enhances the depressor effect of AC and that this effect is counteracted by blocking nitric oxide synthesis and by P. The link between C and P and the nitric oxide pathway needs further exploration. Supported by AUB.
Simvastatin (Zocor®), but not atorvastatin (Lipitor®) or lovastatin (Mevacor®), impairs G2/M cell cycle transit in myeloid cells
Authors: Henry Matthew K. , Stockton Sarah , Laughlin Ryan , Glisic Aleksandra , Giles Jennifer
Published: June 2021

The inhibition of HMG‐CoA reductase with statin drugs such as simvastatin, atorvastatin, and lovastatin can disrupt cell survival and proliferation of multiple cell types. Therefore, statins alone or in combination with other agents present an interesting strategy for the treatment of hyper‐proliferative disorders. Here we are interested in the anti‐proliferative effects of the statins in myeloid cells. Myeloid cells require growth promoting cytokines, such as interleukin‐3, for cell proliferation. These cytokines mediate their activity through the activation of several signaling pathways. Previously, we have shown that signaling through Akt is important for efficient cell cycle transit in myeloid cells. Culturing myeloid cells in simvastatin, atorvastatin, or lovastatin disrupts cytokine‐dependent cell cycle transit through the G1 phase of the cell cycle. However, only treatment of cultures with simvastatin results in delayed transit through G2/M phases of the cell cycle. Additionally, only simvastatin decreases protein expression of Akt. Although the statins are effective at inhibiting HMG‐CoA reductase activity, they appear to have varying effects on cell cycle transit in myeloid cells. The characterization of unique pharmacological properties of statins may help identify new therapeutic strategies effective for the treatment of myeloproliferative diseases.

(Iowa Science Foundation – #ISF‐07‐07)
EXPLORING PROTEIN‐PROTEIN INTERACTIONS IN METAL ION TRANSFERS BETWEEN ENZYMES INVOLVED IN MERCURY DETOXIFICATION
Authors: Miller Susan M. , Feng Xiaohua , Harwood Ian , Hong Baoyu , Nauss Rachel
Published: June 2021

In the major bacterial pathway for mercury resistance, Hg(II) and organomercurials [RHg(I)] undergo facilitated entry into the cytoplasm via an integral membrane transport protein (MerT) and are “detoxified” there by the concerted effort of two enzymes, organomercurial lyase (MerB), which catalyzes cleavage of RHg(I) to Hg(II) and a hydrocarbon, and mercuric ion reductase (MerA), which catalyzes reduction of Hg(II) to Hg(0) as the ultimate detoxification. With the cytoplasmic location of these enzymes and the high toxicity of RHg(I) and Hg(II) due to their affinity for cellular thiols, it is anticipated that proteins in the pathway would have evolved to transfer Hg(II) through direct protein‐protein interactions (MerT‐MerA and MerB‐MerA). Although appealing, evidence supporting this hypothesis is limited. Our current goal is to elucidate the extent to which protein‐protein interactions play a role in metal ion transfers between proteins in the pathway and to understand what type of complexes have evolved to provide the most efficient handling of metal ion for rapid detoxification. Key questions include which protein domains interact and whether they form thermodynamically stable complexes or interact transiently during metal ion transfers. Studies characterizing key players involved in interactions between these proteins will be discussed.
Cardiac P2X4 Receptor Overexpression Can Prolong Survival After Infarction and Rescue Ischemic Heart Failure
Authors: Sonin Dmitry , Zhou Siyuan , Sonina Tatiana , Cronin Chunxia , Liang Bruce
Published: June 2021

Cardiac P2X purinergic receptors (P2XR) can mediate an increased contractility and a potentially important role in the heart. The objective here was to study its role in post‐infarct ischemic heart failure. The P2X4 receptor is an important subunit of the native cardiac P2XR and its cardiac‐specific transgenic overexpression (TG) was developed as a model. At 1 month after infarction (MI), WT mice showed impaired fractional shortening (FS) by echocardiography (18.5 ± 0.5%, SE, n=33 mice, vs. pre‐ligation: 33.7 ± 1.7%, n=8 mice, P<0.05) and +dP/dt (3589 ±142 mmHg/sec vs. sham: 5360 ± 303 mmHg/sec, n=8 mice, P<0.05) by isolated working heart model when compared to sham‐ or non‐operated mice. As early as 7 days after MI, TG animals showed a greater FS (22.6 ± 0.8%, n=18 TG female vs. 20.4 ± 0.6% WT female, P<0.05; 24.5 ± 0.95%, n=20 TG male vs. 17.6 ± 0.5% WT male, P<0.05) and an improved +dP/dt and LV developed pressure. The enhanced contractile function of TG hearts was evident at 1 month after MI with a greater level of FS (23.1 ± 0.7%, n=17 TG female vs. 18.5 ± 0.5%, n=22 WT female, P<0.05; 20.4±0.9%, n=TG male vs.17±0.9%, WT male, P<0.05), of +dP/dt (4370 ± 168 mmHg/sec in TG vs. 3589 ± 142 mmHg/sec in WT, P<0.01), and of cardiac output. The enhanced contractile performance was sustained 2 months after MI, and was associated with increased survival of the TG animals at 2 months. The improved survival was due primarily to decreased deaths during the first 7–10 days after MI. The P2XR represents a novel pathway capable of rescuing ischemic heart failure.
Protective effect of the hydrogen sulfide donor IK‐1001 in a rat model of myocardial ischemia‐reperfusion injury: comparison with ischemic preconditioning
Authors: Su Chienmin Frank , Szabo Csaba , Toombs Christopher
Published: June 2021

In this study, we optimized the timing regiment of IK‐1001, a novel parenteral H2S donor required for cardioprotection in a rat model of myocardial ischemia reperfusion injury. Myocardial ischemia/reperfusion was produced in anesthetized and ventilated male Sprague‐Dawley rats by tightening a ligature on the left main coronary artery for 30 min then releasing it, followed by overnight reperfusion and recovery. Animals were randomized to receive intravenous IK‐1001 or vehicle at various time point; or ischemic preconditioning (IPC: 3x, 5 min I/R) as a positive control. Infarct size was determined by planimetry using TTC. Similar to effect of preconditioning, IK‐1001 (3.85 mg/kg) injected just prior to the ischemia reduced the infarct size by 63% and no difference in AAR. Less protection was found when IK was administered delayed at later phase of ischemia (1.5 min before reperfusion) and early reperfusion (1.5 min after reperfusion). Combining the treatment with hydrogen sulfide donor with IPC failed to show additional benefit as compared to preconditioning alone. Thus, prophylactic treatment of the novel sulfide donor IK‐1001 or initiating sulfide therapy at the time just prior to reperfusion results in infarct size reduction in a setting of myocardial I/R. Moreover, the benefit does not interfere or surpass the IPC protection.
Urinary protein/peptide adducts as markers of reactive naphthalene (NA) metabolite formation in male mice
Authors: Pham Nathalie , Buckpitt Alan , Morin Dexter , Shields Christina DeStefano
Published: June 2021

NA, an abundant PAH generated during combustion, has been shown to produce dose dependent cytotoxicity in murine airways and in rat and murine nasal epithelium. The relevance of findings in these animal models to human health is not clear, in part due to a high background of human lung diseases. Previous work has shown metabolism of NA in target tissues of rodents to reactive metabolites that covalently bind to such proteins as redox, extracellular, and cytoskeletal (Chem Res.Tox 18: 802, 2005). The current work focuses on monitoring urinary protein/peptide adducts as markers of key processes associated with NA toxicity. 24‐hour urine collected from male mice following ip administration of 14C‐NA revealed specific activities of covalently adducted proteins that exceed those observed in lung. On SDS PAGE gels, these adducted proteins varied in size from 5–70 kDa. Much of the adducted protein in male mouse urine was approximately 12 kDa and is consistent with the formation of adducts with major urinary protein (MUP). Female mice, lacking MUPs, have significantly less protein which was adducted at lower specific activity in the urine. Resolution of urinary proteins by 2D electrophoresis and their subsequent identification by mass spectrometry is ongoing. We conclude that adducted proteins eliminated in the urine may be excellent markers of processes critical to target tissue toxicity. Supported by NIEHS 04311 and 04699.
Induction of human and murine hepatic hydroxysteroid sulfotransferase gene expression by RNA interference‐mediated knock‐down of PAPS synthase 2
Authors: Fu Jiaqi , Fang Hai‐Lin , A Thomas
Published: June 2021

Hydroxysteroid sulfotransferase (SULT2A) catalyzes the transfer of sulfate from the physiological sulfate donor 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) to endogenous and xenobiotic substrates. In human hepatocytes, PAPS synthesis is catalyzed by PAPS synthase 2 (PAPSS2). HepG2 cells were stably transduced with lentivirus expressing either non‐targeting small interfering RNA (siRNA) (siNT‐HepG2) or siRNA directed against PAPS synthase 2 (siPAPSS2‐HepG2). Relative to siNT‐HepG2, siPAPSS2‐HepG2 cells transiently transfected with a reporter plasmid containing 1.5 kb of a murine SULT2A 5′‐flanking region demonstrated a significant (~2‐fold) increase in reporter expression. Disruption of a putative LXR motif in the murine SULT2A 5′‐flanking region suppressed the induction of SULT2A reporter expression produced by PAPSS2 knock‐down. Real‐time RT‐PCR analysis also demonstrated ~3‐fold induction of endogenous human SULT2A1 mRNA expression by PAPSS2 knock‐down. These results indicate that both human and murine SULT2A transcription are up‐regulated in response to PAPSS2 knock‐down, and implicate a role for LXR as a sulfate sensor. Supported by ES058223 (M.R.M.), HL50710 (T.A.K) and ES06636.
Phosphodiesterase 7B gene promoter polymorphism in patients with chronic lymphocytic leukemia.
Authors: Peiro Ana M , Tang Chih‐Min , Chou Daisy , Rassenti Laura , Murray Fiona , Zang Lingzhi , Kipps Thomas A , Insel Paul A
Published: June 2021

We have found that B‐cell chronic lymphocytic leukemia (CLL) have a several‐fold increase in the mRNA of phosphodiesterase 7B (PDE7B), which catalyzes cAMP hydroysis. In order to assess the molecular basis for this increase, we tested for the presence of single nucleotide polymorphisms (SNPs) in the promoter and coding regions of the gene (chromosome 6q23‐q24). We thus undertook SNP discovery and expression using DNA isolated from peripheral blood mononuclear cells of 100 control subjects and 151 CLL patients and analyzed differences by Chi square testing. We didn't identified any SNPs in coding region but one in non‐coding, −347G>A. This SNP had a similar frequency in CLL (3%) and controls (2%, P = 0.4669); however, it was found more frequently in males vs. females. A potential explanation for this gender difference is that position −347 is a putative binding site for SRY (sex‐determining region of the Yp11.3 chromosome), which encodes a transcriptional factor that initiates male sex determination and thus, may be related to the predisposition of males to CLL. Using a luciferase assay reporter, we found that −357G>A significantly lowered the promoter activity compared to wildtype (p<0.05). Thus, the presence of −357G>A SNP in the promoter region of PDE7B, although not associated with CLL, can alter promoter activity and thus may contribute to inter‐individual and gender differences in PDE7B expression and activity.
Dysregulation of intestinal CYP3A4‐dependent 1,25‐dihydroxyvitamin D3 catabolism: a potential mechanism for drug‐induced osteomalacia
Authors: Zheng Emily Xi , Zhou Changcheng , Xu Yang , Watkins Paul B. , Assem Mahfoud , Schuetz Erin G. , Thummel Kenneth E.
Published: June 2021

Long‐term therapy with some anti‐epileptic drugs, especially hPXR ligands, has been reported as a risk factor for osteomalacia. However, the mechanism has not been fully elucidated. In this study, we investigated the change of intestinal expression of CYP3A4, CYP24, TRPV6 (the gate‐keeper in calcium absorption) with 1,25(OH)2D3 and/or rifampin treatment in cultured LS‐180 cells and in vivo. Treatment of the cells with 1,25(OH)2D3 caused a 20‐fold induction of TRPV6 expression. Moreover, we observed that a 20‐fold increase in CYP3A4 expression with rifampin pre‐treatment, prior to 1,25(OH)2D3 treatment, was associated with a 20% decrease in TRPV6 mRNA expression, compared to cells treated with 1,25(OH)2D3 alone. In vivo, duodenal biopsy samples from 5 healthy volunteers treated with rifampin exhibited similar results. There was a 6‐fold increase in the duodenal CYP3A4 mRNA level and a 30% decrease in TRPV6 mRNA expression when subjects received rifampin (150 mg, q6h) for 14 days. In contrast, no significant change in the CYP24 mRNA level was detected after rifampin treatment both in vitro and in vivo. These findings suggest that an induction of CYP3A4 in human small intestine by hPXR ligand treatment might result in impaired calcium absorption, and therefore predispose individuals to bone demineralization and osteomalacia. Supported in part by grants from NIH: GM63666, ES07033, GM60364, GM38149 and RR000046.
LC‐MS/MS Method for Probe Drugs in Human Plasma
Authors: Reed Gregory A , Gor Parul , Alnouti Yazen
Published: June 2021

A liquid chromatography‐tandem mass spectrometry method has been developed and validated for the simultaneous determination of probe drugs and their metabolites in human plasma. The analytes include omeprazole and its metabolites omeprazole sulfone and 5‐hydroxyomeprazole; buspirone and its metabolite 1‐ [2‐pyrimidyl] piperazine (1PP); and fexofenadine. These analytes provide indices for the activities of CYPC19, CYP3A4, and OATP1A2. These substrates and metabolites, along with the internal standard lansoprazole, are isolated from plasma using a protein precipitation with acetonitrile. Chromatography was performed using a Luna C18 column eluted with a gradient of 7.5 mM ammonium bicarbonate (pH 8) and acetonitrile. The mass spectrometer was operated in positive ion electrospray mode and multiple reaction monitoring was used for detection and quantification. The method was validated to quantify the concentration ranges 1.0–1000 ng/ml for omeprazole, omeprazole sulfone, 5‐hydroxyomeprazole and fexofenadine, 0.05–100 ng/mL for buspirone, 1.0–100 ng/mL for 1PP. The precision was between 1.2 – 8.2%, and the accuracy ranged from 85.6 – 114% for these analytes. The lower limit of quantification for buspirone was 0.05 ng/mL; whereas the LLOQ for all other analytes was 1 ng/mL. This method provides a fast and highly sensitive analytical tool for quantification of the key analytes from this probe drug cocktail.
Induction of cellular stress via the p38 MAPK pathway drives VELCADE® (bortezomib)‐induced apoptosis in esophageal squamous cell carcinoma cells
Authors: Lioni Mercedes Ines , Noma Kazuhiro , Herlyn Meenhard , Smalley Keiran
Published: June 2021

Esophageal carcinoma is known to have a 5‐year survival rate of less than 20%. This shows a failure of current treatment regimens to provide patients with a significant life expectancy after diagnosis.

In preliminary studies we used a novel three‐dimensional (3D) model, formed by esophageal squamous cell carcinoma (ESCC) cell aggregates embedded into collagen type I to allow growth and invasion within the 3D tissue like structure. Treatment with bortezomib, a proteasome inhibitor, was strongly growth inhibitory in both conventional and 3D settings. In a 3D reconstruction model of a primary esophageal tumor, TUNEL staining revealed the induction of apoptosis using 0.5uM of the drug. Induction of apoptosis proceeded through activation of caspase‐9 and ‐3, and was p53 independent. Animal experiments were performed using TE12 ESCC xenografts in NOD/SCID mice. Treated animals showed a bortezomib‐specific reduction of tumor size associated with decreased Ki67 staining and increased TUNEL positivity. Proteasome inhibitors are known to induce cellular stress. Treatment of the ESCC lines with bortezomib led to increased activity in both the p38 MAPK and JNK pathways. A role for p38 MAPK in bortezomib‐induced apoptosis was demonstrated by the ability of the p38 inhibitor SB230580 (10uM) to block the bortezomib‐induced cleavage of caspase‐3. Identical results were observed using selective knockdown of p38 MAPK using siRNA. Our investigation suggests that p38 MAPK induction could be a useful strategy for apoptosis induction when combined with bortezomib.

Supported by the National Cancer Institute (grant P01‐CA098101)
Characterization of a drug resistant phenotype in chronic myeloid leukemia
Authors: Nichols Jennifer , Tanaka Hideo , Christopherson Richard , Graves Lee
Published: June 2021

Philadelphia‐positive chronic myeloid leukemia (CML) is a cancer identified by the fusion of BCR with the ABL kinase resulting in a constitutively active kinase and uncontrolled cell growth. Gleevec (STI‐571) is a kinase inhibitor targeting BCR‐ABL and is currently the most effective therapy for CML. A significant fraction of CML patients develops drug resistance to Gleevec, and multiple mechanisms of resistance have been described. Recently, a cell line, MYL, was developed as a model of CML along with its Gleevec‐resistant counterpart (MYLR). We used a novel antibody microarray system (Medsaic) and immunoblotting to examine cell surface and protein expression differences in MYL and MYLR cells. Increased levels of tissue transglutaminase, c‐Kit, CD44, CD31, and CD11a were observed in MYLR cells, all of which have previously been shown to have an important role in cell survival and proliferation. Identification of these differences that characterize the drug resistant phenotype has critical implications to further elucidating the direct mechanisms of drug resistance as well as providing new targets for effective therapies. Funding for this research has been provided by NIEHS Training Grant T32‐ES07126, NIH R01‐GM069976, and the PhRMA Foundation.
Cytolytic peptides on nanoparticle carriers induce dramatic melanoma tumor shrinkage in vivo by apoptosis
Authors: Soman Neelesh , Lanza Gregory , Schlesinger Paul , Wickline Samuel
Published: June 2021

The potential application of cytolytic peptides (CP) for cancer therapeutics has met with limited success due to in vivo degradation and systemic toxicity. We now illustrate the first successful in vivo deployment of a “first‐generation” CP (melittin, a 26 amino‐acid amphipathic peptide in bee venom) for cancer therapy after incorporation onto a lipid monolayer of a perfluorocarbon (PFC) nanoparticle (NP, size ~200 nm). The hydrophobic brominated PFC core maintains the structural integrity of the NP (as shown by electron microscopy) and allows a very slow dissociation of melittin (Kd ~1.5 nM by surface plasmon resonance). In vitro, melittin‐NPs are effective against C‐32 melanoma cells (IC50 ~ 32 μM) and cause a non‐porative apoptotic cell death as revealed by annexin‐FITC/propidium iodide staining and cytochrome c/lactate dehydrogenase release assays. Although free melittin is toxic in vivo (LD50 ~ 4 mg/kg), four doses of melittin‐NPs (8 mg/kg) are effective in causing a 7.6‐fold reduction in the volume of B16 melanoma tumors in C57BL/6 mice (n=5, p = 0.004) without any associated systemic toxicity. PFC nanoparticles thus represent the first class of unique lipid‐based nanovehicles that can provide a stable and safe synthetic delivery vector for melittin (or other CPs) in vivo by preventing their degradation, reducing systemic toxicity, while simultaneously maintaining effectiveness for cancer therapeutics.
Two fluoroquinolone‐bisphosphonate (BP) derivatives show similar binding affinity to calcium phosphate (CP) particles, but have different antibacterial activity
Authors: Buxton Thomas Brooks , Runner Royce R , Herczegh Pal , McPherson James C
Published: June 2021

We previously demonstrated that osteoadsorptive BP conjugates of fluoroquinolones (E41, ciprofloxacin‐BP), carried on CP particles (μ‐sized Skelite™), packed into a traumatized bony defect, showed value in the treatment of rat tibial osteomyelitis (British J Surg, 2004;91:1192–1196). A Gatifloxacin‐BP conjugate (GB) was contrasted to E41 for activity against methicillin‐susceptible Staphylococcus aureus, and other pathogens. Minimum inhibitory concentration (MIC) and Mininum Bactericidal Concentrations (MBC) were determined. In vitro binding studies, holding levels of E41 and GB constant (2 μg/ml), while increasing levels of CP (nm‐sized NanOss™), were determined by measurement of fluoroquinolone‐BP associated fluorescence. GB showed greater MIC and MBC activity against several Gram‐positive and Gram‐negative pathogens, e.g., an 8‐fold difference with S. aureus (Table). Binding appeared to be saturable. Maximal binding was: E41, 89.7 % ± 4.74, vs. GB, 76.8 % ± 2.35 (p≤0.05). The dissociation constant was: E41, 1.35 × 10−8 mM (NanOss™), vs GB, 1.83 × 10−8 mM (p≤0.05).

Versus E41, GB has (i) improved activity against bone‐pathogenic S. aureus, and other bacteria, and (ii) shows a similar binding affinity to NanOss™, used as a CP vehicle for topical drug delivery.
Lipid peroxidation in Tradescantia pallida: a new bioindicator response of air pollutants
Authors: Rhoden Claudia Ramos , Carneiro Maria Fernanda Hornos , Petry Marcelo Rafael , Oliveira Regiani Carvalho , Saldiva Paulo Hilário Nascimento
Published: June 2021

Tradescantia pallida is a very common garden plant which is widely used as a bioindicator. The aim of this study was to test this plant as a model to detect oxidative damage as well as genotoxicity and accumulation of trace elements induced by vehicular emissions. Twenty vases of Tradescantia pallida were distributed into two sites: 1) rural area=control; 2) urban area, for 2 months during the winter. We evaluated the genotoxicity (by the frequency of micronuclei (MCN) in meiotic pollen mother cells), the accumulation of trace elements in leaves ( by X‐Ray Flourescence Analysis) and the oxidative damage (by malondialdehyde ‐MDA‐ concentration) in leaves. We found an increase of MCN frequency in the urban area site as compared with the rural area (%MCN – rural area = 0.73+/−0.49; urban area= 4.61+/−1.17; p<0.05) as well as an increase of MDA ([MDA]: micromol/fw ‐ rural area= 0.018+/−0.001 X urban area= 0.022+/−0.001; p=0.023). The rural area presented lower concentration of Br, Ca, Cl, Co, Mg, Mn, Mo, Pb, Sc and Zn as compared to the urban area. Our study showed that Tradescantia pallida serves both as a bioindicator of genotoxic substances as of oxidizing contaminants. In addition it is capable of accumulating trace elements in their leaf tissue.

Support: CNPq, FFFCMPA, Secretaria da Saúde do Esatdo do Rio Grande do Sul ‐ Brazil
Preconditioning by tissue bath reveals differential effects based on location within an arteriolar network.
Authors: Uddin Sardar Zia , Frame Mary D
Published: June 2021

Pre‐exposure to L‐arginine (L‐arg, micropipette) preconditions individual arteriolar networks by 15 minutes after the pre‐exposure interval. Preconditioning is seen as a change in response to L‐arg from constriction to dilation (Microcirculation, 2007:14, 739–751). We hypothesized that tissue bath pre‐exposure to L‐arg would precondition all arterioles uniformly at a common threshold concentration. The cheek pouch of anesthetized (70 mg/kg pentobarbital) male hamsters (n=10) was prepared for intravital microscopy. Diameter change was measured as a function of L‐arg (5 min, 10–11‐ 10‐7M), either without preconditioning (PC, control) or with PC (15 min of L‐arg 10–11‐ 10‐7M) followed by a 15 min waiting period. Data was obtained from each sequential branch point of the network (1st, 2nd, 3rd, last), and at 3 segments in each bifurcation (Feed, Feed continuation, Branch). At low L‐arg, all arterioles constricted ± PC. The dose of L‐arg to PC all networks (Initiation Threshold) was ∼10‐7M. Separately, we examined the test dose of L‐arg where dilation was first seen (Experimental Threshold, ET). ET did not vary by branch vs. feed designation. ET varied along the length of network; ET upstream was ∼ 10‐7M while downstream was lower (∼10‐10M). We conclude that network position predicts the extent of altered arteriolar vasoactive responses for tissue bath preconditioning. (AHA 0655908T)
Species differences in collaterals arising from femoral artery occlusion: a comparison from mice to men
Authors: Distasi Matthew R , Ziegler Matthew A , Case Jamie , Miller Steven J , Murphy Michael P , Dalsing Michael C , Sturek Michael , Unthank Joseph L
Published: June 2021

Animal studies of therapies to enhance vascular compensation to arterial occlusion have failed to predict clinical outcomes, indicating the need for improved models. Collateral arteries resulting from femoral artery occlusion were compared between humans, mice, rats, and pigs. Collateral location and size was determined from angiograms and vascular casts. Wall morphology was assessed from cross‐sections in all species except human. The primary collaterals orginated and re‐inserted in non‐ischemic upper leg tissues. Dramatic intimal cell proliferation/recruitment occurred; the internal elastic lamina remained intact without evidence of neo‐intimal formation. Collateral size ranged from ∼0.1 mm in mice to >1 mm in larger species. A vasa vasorum was not observed in mouse collaterals and was much more extensive in pig than rat collaterals. A 10–100‐fold difference was observed between species in vascular wall thickness, ratio of endothelial to smooth muscle cells, and distance from ischemic tissue. These differences would be expected to impact the balance between growth modulators and reactive species and their paracrine effects due to diffusion distances. In addition, it is unknown how differences in hemodynamics and the presence of a vaso vasorum may impact collateral growth. Studies are warranted in larger species which may more closely mimic human collaterals. Support: HL42898, RR013223, HL062552.
Elevated periarterial hydrogen peroxide in resistance vessels of spontaneously hypertensive rat (SHR) during rest and increased flow is reversed by apocynin
Authors: Zhou Xiaosun , Bohlen H Glenn , Miller Steven J , Unthank Joseph L
Published: June 2021

Flow‐mediated dilation is mediated by H2O2 in humans with arterial disease. We have shown that NO does not increase with elevated flow in resistance arteries of SHR and aged rats. These same vessels do not enlarge as collaterals. We tested the hypothesis that flow‐mediated H2O2 is upregulated in SHR, and reversed by the NADPH oxidase inhibitor, apocynin. Our mesenteric model was used in young WKY rats, SHR and SHR pre‐treated with apocynin (Apo‐SHR). H2O2‐specific electrodes were used to measure real‐time, in vivo mesenteric periarterial [H2O2]. Specificity of electrodes was verified with catalase. After H2O2 baseline measurement, collateral flow was increased 50–200 % by clamping adjacent arteries. Baseline H2O2 was significantly increased in SHR compared with WKY (26.2 ± 6.8 vs. 7.2 ± 0.9 μM, p< 0.05) or Apo‐SHR (11.1 ± 3.2 μM). Successive increases in flow significantly increased periarterial [H2O2] in SHR from baseline (1 clamp, 46 ± 16 %, p<0.05; 2 clamps, 61 ± 11 %, p<0.05). Flow‐mediated [H2O2] increases were prevented in Apo‐SHR (−18 ± 5 %, 6 ± 23 %; 1 and 2 clamps respectively) and not observed in WKY. The results indicate that elevated baseline and flow‐mediated H2O2 production may originate from NADPH oxidase in SHR. Flow‐mediated H2O2 production may be a compensatory mechanism for vasodilation in SHR but may also be responsible for collateral growth impairment.

Support: HL42898 and HL20605
Regulation of local angiotensin type 1b receptor (AT1bR) expression explains collateral growth impairment in spontaneously hypertensive rats (SHR) and restoration of collateral growth (CG) by captopril
Authors: Miller Steven J. , Zhou Xiaosun S. , Norton Laura E. , Murphy Michael P. , Dalsing Michael C. , Bohlen H. Glenn , Unthank Joseph L.
Published: June 2021

The SHR is characterized by an activated RAS, increased oxidative stress, endothelial dysfunction and impaired CG. Oligonucleotide microarray analyses were performed with SHR and WKY to gain insight into the molecular basis of CG impairment. The results demonstrated a fundamental difference in the overall gene expression pattern in collateral arteries compared to same animal controls. Downregulated collateral genes included AT1bR and angiotensinogen (AGT) while p22phox was upregulated in SHR control arteries relative to WKY (2.14+/− 0.50 vs 0.93+/−0.10). RT‐PCR confirmed the suppression of AGT in SHR and WKY (56+/−29% and 40+/−9%) and of AT1bR in SHR (42+/−9%). However, the AT1bR was upregulated in WKY collaterals (302+/−70%). Pretreatment with captopril, which we have previously shown to restore CG (Miller, AJP‐Heart, 292:2007), restored flow‐mediated NO production as assessed by in vivo periarterial NO measurement, prevented AT1R downregulation in collaterals (174+/−61%), and decreased p22phox expression. Losartan had no effect in SHR but prevented CG in WKY. Apocynin restored CG in SHR and had no effect in WKY. The results indicate an essential role for the AT1R in CG and suggest that the transcriptional regulation of this molecule during CG is modulated by the balance between oxidative stress and nitric oxide.

Support: HL42898 and HL20605.
Immune‐mediated vasomotor dysfunction and ROS production during hypercholesterolemia occurs through an IFN‐γ‐dependent signaling pathway
Authors: Wolfort Ryan Michael , Stokes Karen Y , Granger D Neil
Published: June 2021

Hypercholesterolemia (HC), a risk factor for the development of atherosclerosis, elicits a systemic inflammatory response that is accompanied by endothelium‐dependent vasomotor dysfunction. Although immune‐derived cytokines and the production of reactive oxygen species (ROS) have been implicated in atherogenesis, it remains unclear how these factors contribute to HC‐induced vasomotor dysfunction. We tested the hypothesis that HC elicits immune‐mediated ROS production and endothelial dysfunction through IFN‐γ signaling. Wire myography was employed to assess vasomotor responses and a cytochrome C reduction assay was used to assess ROS production in aortic rings harvested from wild type (WT) or mutant mice placed on either a normal (ND) or cholesterol‐enriched (HC) diet. Bone marrow chimeras (BMC) were produced by marrow transplantation between WT and IFN‐γ‐deficient (IFN‐γ−/−) mice. Endothelium‐dependent vasodilation to Ach was impaired and ROS production was increased in WT and WT→WT BMC mice on HC. The impaired vasodilation and increased ROS production were absent in IFN‐γ−/−, IFN‐γ−/−→WT BMC, and IFN‐γ receptor‐deficient mice on HC diet. Our findings indicate that immune‐mediated endothelium‐dependent vasomotor dysfunction and increased ROS production caused by HC occurs through an IFN‐γ signaling pathway.
Involvement of MicroRNA‐21 in vascular smooth muscle cell migration reveals a novel mechanism in cell motility in proliferative vascular disease
Authors: Zhang Chunxiang , Cheng Yunhui , Liu Xiaojun , Lin Ying , Yang Jian
Published: June 2021

MicroRNAs (miRNAs) are a recently discovered class of endogenous, small, noncoding RNAs that regulate gene expression by either translational inhibition and / or mRNA degradation. Our recent study suggests that miRNAs are aberrantly expressed in rat carotid arteries with neointimal lesion formation. Among them, MicroRNA‐21 (miR‐21) is the most upregulated miRNA that had more than a 5‐fold increase compared with the control. Vascular smooth muscle cell (VSMC) migration plays an important role in proliferative vascular diseases such as atherosclerosis and restenosis after angioplasty. The aim of the current study is to determine the potential role of miR‐21 in VSMC motility. In vitro, miR‐21 expressed was significantly increased in cultured rat VSMCs stimulated by PDGF. Knockdown of miR‐21 expression decreased, whereas overexpression via adenovirus‐mediated gene transfer increased PDGF‐mediated VSMC motility. miR‐21 expression in VSMCs isolated from balloon‐injured vessels is significantly higher than that from normal vessels. Knockdown of miR‐21 expression decreased, whereas overexpression via adenovirus‐mediated gene transfer increased VSMC migration in rat carotid arteries after angioplasty. The results indicate that miR‐21 is involved in VSMC migration both in vitro and in vivo. MiR‐21 may play an important role in proliferative vascular disease via modulating VSMC motility.
Hyperhomocysteinemia Impairs Agonist‐Induced eNOS Phosphorylation on S1179
Authors: Ashley Blair , Looft‐Wilson Robin
Published: June 2021

Previously, our group found that hyperhomocysteinemia (HHcy) impairs endothelium‐dependent vasodilation in mouse mesenteric arteries, which was attributed to attenuated nitric oxide (NO)‐dependent dilation, and coincident with decreased eNOS expression and decreased basal eNOS phosphorylation of serine 1179 (S1179) (Ashley et al., FASEB J 21:A910, 2007). Phosphorylation of eNOS of S1179 is necessary for NO‐dependent vasodilation in response to acetylcholine (Ach) stimulation. Thus, we hypothesized that HHcy impairs Ach‐induced eNOS phosphorylation at S1179. To test this hypothesis, we stimulated mesenteric arteries, ex vivo, with Ach (10−4 M) for 5 min and compared the ratio of phosphorylated eNOS at S1179 to total eNOS protein in diet‐induced HHcy mice (Hcy: 89.2 ± 49.0 μM) and their age‐matched controls (Hcy: 6.6 ± 1.9 μM). Densitometric quantification of protein immunoblots indicated robust S1179 phosphorylation in response to Ach in control arteries (% increase ± SD: 59.4% ± 18.5%), but not in HHcy arteries (4.8% ± 18.0%), a response that was significantly less in HHcy arteries (P<0.05). We conclude that attenuation of agonist‐induced NO‐dependent vasodilation in mesenteric arteries from mice with chronic HHcy, is at least partially caused by impaired S1179 phosphorylation of eNOS, and therefore reduced activation of eNOS. Supported by the American Heart Association and the American Physiological Society Undergraduate Summer Research Fellowship.
Connexin phosphorylation at the myoendothelial junction can regulate intercellular calcium communication
Authors: Isakson Brant Edward , Best Angela , Robert M
Published: June 2021

The myoendothelial junction (MEJ) is a specialized region where endothelial cells (EC) and smooth muscle cells (SMC) make contact. We have previously demonstrated polarized IP3 responses between EC and SMC across the MEJ and hypothesized that the polarized IP3 movement may be due to connexin phosphorylation. Because Cx43 was localized to the mouse MEJ in situ, and the C‐terminus of Cx43 is highly phosphorylated, we focused on serine368 (Cx43‐S368) which is known to mediate gap junction closing via protein kinase C activation. As a marker for the MEJ in situ, we used phalloiden to mark actin bridges linking EC and SMC and demonstrate Cx43‐S368 staining. In vitro, primary cultures of mouse cremasteric EC and SMC were placed in a vascular cell co‐culture with Cx43 at the MEJ. Stimulation of SMC produced a global increase in EC intracellular Ca2+ concentrations ([Ca2+]i). Application of the PKCε activator FGF‐2 (10 μg/mL), and stimulation of SMC, did not induce an increase in EC [Ca2+]i, indicating possible closed gap junctions at the MEJ. However confocal line scans demonstrated a small increase in [Ca2+]i in the cellular extensions composing the MEJ, implicating Ca2+ thresholds as insufficient to induce a global increase in [Ca2+]i. We believe that connexin phosphorylation at the MEJ may regulate gap junction opening and closing so as control heterocellular calcium communication. Research supported by NIH and AHA.