bluuebunny/crossref_metadata_2025_split huggingface dataset:
Coordinate Dysregulation of Multiple MicroRNAs that Regulate DNMT3b Expression Characterizes A Subset of Basal‐like Breast Cancers
Authors: Sandhu Rupninder , Livasy Chad A , Coleman William B 
Published: June 2021

Basal‐like breast cancers frequently express a hypermethylation defect associated with DNMT hyperactivity and DNMT3b overexpression. To determine the mechanism governing DNMT3b overexpression, we examined the expression of microRNAs (miRs) that regulate DNMT3b (miR‐29a, 29b, 29c, 148a, 148b) or are predicted to regulate DNMT3b (26a, 26b, 203, 222) by qPCR (normalized to RNU66) in 70 primary breast cancers (36 luminal A, 13 luminal B, 5 Her2+, 16 basal‐like) and 18 normal breast tissues. Significantly reduced expression of miR‐29c distinguished basal‐like breast cancers from other subgroups. MiR expression patterns revealed two groups among basal‐like breast cancers corresponding to low expression (n=10) and high expression (n=6). Average miR expression was significantly reduced in low vs. high groups (4.4 to 22.2‐fold difference; p<0.05). 50% of basal‐like tumors showed reduced expression of 7/9 miRs compared to 22% of luminal A, 15% of luminal B and 20% of HER2+ tumors. These results suggest strongly that (i) reduced expression of 29c is characteristic of basal‐like breast cancers, (ii) two subgroups of basal‐like breast cancers can be identified based on miR expression patterns, and (iii) subgroup of basal‐like breast cancers with reduced expression of multiple regulatory miRs may correspond to those expressing a hypermethylation defect related to DNMT3b overexpression.UNC University Cancer Research Fund
Prion Disease Prevention: Mapping Genetic Transmission Etiology
Authors: Kelly Amy Catherine , Mateus‐Pinilla Nohra , Douglas Marlis , Douglas Michael , Ruiz Marilyn , Brown William , Beissel Tom , Shelton Paul , Novakofski Jan 
Published: June 2021

Transmission of bovine spongiform encephalopathy (BSE) from cattle to humans (as variant Creutzfeldt‐Jakob disease) demonstrates zoonotic mechanisms in the transmission of infective prions. We had two aims: 1) to better understand molecular elements implicated in prion disease transmission and 2) to predict spread of Chronic Wasting Disease (CWD) by quantifying gene flow from infected to uninfected locations. Deer were used as a model species due to high transmission prevalence of CWD which, unlike BSE, appears to spread horizontally and indirectly through the environment. Prion gene (Prnp) mutations in deer were investigated using gene sequencing. Four alleles were negatively and one positively associated with CWD. Cumulative mutation number lowered risk of infection by 50% per mutant allele.To derive genetic interconnectivity, we juxtaposed molecular genetic profiles with landscape features that could impede population linkages. Males were identified as long‐distance disease carriers (movements of ~100 km) compared to females (movements of ~40 km). Counter‐intuitively, forest fragmentation promoted movement, potentially increasing human and livestock exposure to diseased animals. Our results offer a potential tool for evaluating the relationship between prion translation, folding and disease as well as the synergistic interplay of genotype, sociobiology, and environment. [USFWS, IDNR]
PKC β inhibitor ruboxistaurin prevents the increase of 15‐F2t‐isoprostane in the myocardium and plasma in Type 1 diabetic rats
Authors: Liu Yanan , Lei Shaoqing , Liu Hui‐min , Mao Xiaowen , Wong Gordon T , Vanhoutte Paul M , Irwin Michael G , Xia Zhengyuan 
Published: June 2021

Levels of 15‐F2t‐isoprostane (IsoP), a specific marker of oxidative stress and a recently identified independent predictor of myocardial morbidity in patients with ischemic heart disease, are increased in the myocardium and plasma in Type 1 diabetes. The beta isoform of Protein kinase C (PKCβ) is over‐expressed in the myocardium of diabetic rodents and contributes to the development of diabetic cardiomyopathy and associated complications. We postulated that selective PKCâ inhibition with ruboxistaurin would attenuate hyperglycemia‐induced oxidative stress and normalize IsoP production. Control or streptozotozin‐induced diabetic rats were treated (DT) or untreated (C, D) with ruboxistaurin (1 mg/kg/day) delivered by oral gavage for four weeks. Myocardial IsoP content, cardiac mass and plasma IsoP were significantly increased while there was a significant decrease in superoxide dismutase activity in D compared to C rats (all P<0.05). Ruboxistaurin treatment normalized all these changes. It is concluded that ruboxistaurin may have prevented hyperglycemia‐induced oxidative stress by restoring endogenous antioxidant enzyme superoxide dismutase activity in diabetes.Supported by RGC/GRF grants (HKU781109M to ZX, HKU766709M to MGI) and University of Hong Kong seeding grant for basic research (ZX).
Molecular identification of the constitutively‐activated volume‐Regulated Cl‐ channels in hypertrophied ventricular myocytes
Authors: Duan Dayue Darrel , Wang Guanlei , Liu Lihui , Ye Lingyu Linda 
Published: June 2021

The molecular identity of the constitutively‐activated volume‐regulated Cl− current (VRCC) in hypertrophied cardiac myocytes remains elusive. In this study, ClCn3−/− mice were used to test whether ClC‐3 encodes the constitutively‐activated VRCCs. Whole‐cell voltage‐clamp and Western blot were used to examine the changes in VRCC and ClC‐3 expression in left ventricular (LV) myocytes from WT and ClCn3−/− mice subjected to minimally invasive transverse aortic banding (MTAB) for 5 weeks. In most WT MTAB myocytes (76%) a large VRCC was activated under isotonic conditions. Hypotonic cell swelling caused no changes in VRCC while it was significantly inhibited by hypertonic solution. VRCC in WT‐MTAB myocytes had an anion selectivity of I− > Cl−, and was inhibited by tamoxifen, PKC and anti‐ClC‐3 Ab. Expression of ClC‐3 protein in the heart was significantly increased in the WT‐MTAB mice but not in the WT sham‐operated mice. No ClC‐3 expression was detected in the Clcn3−/−‐MTAB hearts. In the age‐matched Clcn3−/−‐MTAB mice, a significantly smaller VRCC (at +80 mV 2.2±0.5 pA/pF, n=5 vs WT‐MTAB 5.4±0.8 pA/pF, n=5, P<0.01) was present in a significantly smaller population of cells (36%, P<0.05 vs WT‐MATB). But VRCC was not increased by hypotonic stress and was not inhibited by tamoxifen, PKC or anti‐ClC‐3 Ab.ConclusionClC‐3 may be responsible for the constitutively‐activated VRCCs in hypertrophied heart. (supported by NCRR P‐20 RR‐15581)
Effects of PETN and GTN on activity of purified human mitochondrial aldehyde dehydrogenase
Authors: Knorr Maike , Daiber Andreas , Kamuf Jens , Pautz Andrea , Oelze Matthias , Art Julia , Kleinert Hartmut 
Published: June 2021

ObjectivePentaerithrityl tetranitrate (PETN) treatment is devoid of nitrate tolerance. In this study we tested whether the beneficial effect is related to the induction of heme oxygenase‐1 or whether PETN may preserve aldehyde dehydrogenase (ALDH‐2) activity in purified enzymatic preparations.Methods/ResultsRecombinant human ALDH‐2 was overexpressed in E. coli using a plasmid: To generate pET16B‐hALDH2, a bacterial expression plasmid coding for a His‐tagged human ALDH2 protein, the plasmid pT7‐7‐hALDH2 (provided by K.K. Ho and H. Weiner, Purdue University, West Lafayette, USA) was digested with NdeI and HindIII. Purity and activity were tested by SDS‐PAGE with Coomassie staining, Western blotting and HPLC‐based activity assay. Reactive oxygen species (ROS) formation was tested by different methods. Glyceryl trinitrate (GTN) caused a severe concentration‐dependent decrease in enzyme activity; PETN‐incubation had only minor effects. Incubation of purified ALDH‐2 with GTN but PETN lead to generation of a reactive species.ConclusionsThis study revealed that PETN has intrinsic antioxidative properties and does not trigger ALDH‐2‐dependent ROS formation from the purified enzyme.
Adolescent nicotine exposure enhances alcohol self‐administration, and subsequently affects extinction of conditioned fear response
Authors: Larraga Armando , McQuown Susan , Belluzzi James D , Leslie Frances M 
Published: June 2021

We have shown that nicotine (Nic) is a ‘gateway’ drug that sensitizes the adolescent brain to other abused drugs. Furthermore, alcohol exposure during development leads to neuroanatomical abnormalities and adverse behavior.We hypothesize that Nic acts as a ‘gateway’ for ethanol (EtOH) self‐administration (SA), and that development of the limbic circuitry can be altered by adolescent Nic and EtOH exposure. We thus tested the effect of brief, low‐dose Nic pretreatment on the acquisition of EtOH SA in adolescent and adult male rats, and then tested for extinction of conditioned fear response (CFR). Adolescent rats, aged postnatal day (P) 28, or adult P86 rats, were given 2 daily intravenous (IV) injections of Nic (0.03 mg/kg/0.1 ml) or saline for 4 consecutive days. At P32 and P90 animals were allowed to IVSA EtOH daily with escalating doses over 10 consecutive days. CFR testing occurred 1 week later.Nic pretreatment enhanced early acquisition of EtOH IVSA in adolescent rats, but not total intake in either age group. Wheareas there was no significant relationship between Nic or EtOH intake and CFR extinction in adult rats, there was an interaction between Nic and EtOH exposure in adolescents, with a significant decrease in CFR with increased EtOH intake. These data suggest that adolescent Nic exposure enhances initial acquisition of EtOH IVSA and that Nic and EtOH have lasting effects on limbic forebrain development.
Regulation of D2 dopamine receptors by G‐protein coupled receptor kinase (GRK) and β‐Arrestin
Authors: Thanawala Vaidehi Jatin , Kovoor Abraham , Celver Jeremy , Sharma Meenakshi 
Published: June 2021

G protein coupled receptor kinase (GRK) and Arrestin (Arr) regulate G protein coupled receptor (GPCR) desensitization and internalization. Our objective was to determine the mechanisms through which GRK and Arr regulate the D2 Dopamine Receptor (D2R) since all current antipsychotic drugs antagonize D2R suggesting that psychosis may arise from aberrant D2R regulation or signaling.It is difficult to distinguish between the effects of GRK and Arrestin on receptor uncoupling and internalization in mammalian cells because both processes occur simultaneously and mammalian cells express high levels of endogenous GRKs and Arrestins. In the Xenopus oocytes expression system, GPCRs do not desensitize unless GRK and Arrestin are exogenously coexpressed. Furthermore, the receptor desensitization that occurs with GRK and Arrestin over expression in Xenopus oocytes is due to uncoupling of the receptor from its effectors and not due to receptor internalization. Thus this system enables the GRK and Arrestin mediated uncoupling, to be studied independently from internalization.We showed that GRK3 and Arr3 coexpression in the Xenopus oocyte system desensitized D2R. We use site directed mutagenesis to identify the regions of the receptor that underlie GRK and Arr‐mediated desensitization and show that the third cytoplasmic loop is an important determinant.
Acute hyperglycemia augments neurovascular injury in diabetes
Authors: Ergul Adviye , Elgebaly Mostafa M , Ogbi Safia , Johnson Maribeth , Mezzetti Erin , Fagan Susan C 
Published: June 2021

BackgroundAdmission hyperglycemia impacts ischemic stroke deleteriously but the relative role of acute hyperglycemia versus diabetes in the pathogenesis of this poor outcome is not clear.PurposeTo test the hypothesis that the impact of acute hyperglycemia on neurovascular damage following ischemic injury is more severe when superimposed on diabetes.MethodsAcute hyperglycemia (HG) was achieved by 3 ml IP glucose injection (300 mM) 20–30 minutes before 3 h middle cerebral artery occlusion (MCAO) in male control Wistar and mildly diabetic Goto‐Kakizaki (GK) rats. The goal was to achieve mild elevation in blood glucose levels (~180 mg/dl) in control rats to mimic glucose levels normally observed in the GK model. Following 21 h reperfusion neurobehavioral tests were performed before sacrifice and neuronal (infarct size) and vascular (hemorrhagic transformation (HT) frequency and excess hemoglobin) damage were measured.ResultsInfarct size was significantly smaller in diabetes. Moderate HG increased neuronal damage in diabetic but not in control rats. HT frequency and hemoglobin was significantly higher in diabetes. HG augmented vascular damage in control rats but had no additional effect on bleeding in diabetes. There was significant neurological deficit in diabetes which was worsened by HG.ConclusionsEven moderate hyperglycemia worsens outcome from ischemic stroke. While moderate HG does not increase infarct size in control rats, it is sufficient to induce HT. A further elevation in blood glucose in diabetic animals does not worsen HT suggesting that diabetic animals already suffer from increased vascular damage.
The Purinergic Agonists ATP and UTP Act Predominantly via P2Y Receptors in Human Vascular Endothelial Cells
Authors: Braun Andrew P , Raqeeb Abdul , Sheng Jianzhong 
Published: June 2021

Purinergic agonists, such as ATP, can produce robust vasodilation, yet the specific receptor(s) (i.e. P2X or P2Y) responsible for these effects remain poorly characterized. In this study, we have examined the purinergic receptors mediating acute Ca2+ mobilization, membrane hyperpolarization and nitric oxide (NO) synthesis in cultured HUVECs (EA.hy926 cell line). Brief application of either ATP or UTP (2 μM) to single HUVECs produced comparable elevations in cytosolic Ca2+, membrane hyperpolarizations and acute NO synthesis. These responses were largely unaffected in the presence of the non‐selective P2X receptor antagonists PPADS (10 μM) and TNP‐ATP (1 μM). In contrast to ATP and UTP, the selective P2X receptor agonist α,β‐methyl ATP (2 μM) had little effect on cytosolic Ca2+, membrane potential or NO production. Collectively, these data suggest that the observed stimulatory actions of ATP and UTP in this cell system occur primarily via P2Y, and not P2X, receptors. A plot of the concentration‐dependent rise in cytosolic Ca2+ evoked by ATP and UTP revealed similar EC50 values for the two agonists (1–3 μM); this equipotent action of ATP and UTP further suggests that P2Y2 receptors are the predominant purinergic sub‐type. Finally, RT‐PCR analysis confirmed the presence of P2Y2 receptor mRNA in this human vascular endothelial cell model.Research support to AP Braun was provided by the CIHR and HSF Alberta
PAR1 inhibition does not enhance the cardioprotective effect of postconditioning
Authors: Jiang Rong , Rutledge Caleb , Shi Weiwei , Guyton Robert A , Vinten‐Johansen Jakob 
Published: June 2021

Postconditioning (postcon) protects the heart from ischemia reperfusion (IR) injury. Protease activated receptor1 (PAR1) inhibition has protective effects after IR. Therefore, we hypothesize that if postcon and pharmacological PAR1 inhibition target the same pathway, and the combination of postcon and PAR1 inhibition may not have additive protective effect.MethodsIn anesthetized open‐chest rats, the left coronary artery (LCA) was occluded for 30 min and reperfused for 3 hours. Rats were randomly assigned to1) Control: saline; 2) PAR1 antagonist (BMS 226001) 1mg/Kg was administered 5 min before R; 3) Postcon with 3 cycles of 10 s R and 10 s re‐occlusion and 4) BMS 1 mg/Kg plus postcon.ResultsArea at risk (36%–39%) was comparable among 4 groups. Compared to control, infarct size (TTC staining) was significantly less in BMS, postcon and the combination groups. The combination of BMS and postcon did not further significantly reduce infarct size compared to either intervention alone.ConclusionsInhibition of PAR1 and postcon alone at reperfusion are cardioprotective in vivo; however, concomitant PAR1 inhibition does not enhance the cardioprotection of postcon.

Group
N
AAR/LV (%)
An/AAR (%)

Control
7
37.9 ± 2.4
57.0 ± 2.1

BMS
8
38.1 ± 2.5
42.5 ± 2.5*

Postcon
7
39.3 ± 2.1
39.1 ± 2.3*

BMS + Postcon
7
35.6 ± 1.4
39.9 ± 3.0*

Values are expressed as mean ± S.E.M.
P < 0.05 vs. values in control.
Effect of Nitric Oxide Synthase and growth conditions on hydrogen peroxide production in cultured endothelial cells during shear stress
Authors: Zinkevich Natalya S , Gutterman David D 
Published: June 2021

Endothelial cells release nitric oxide (NO) and reactive oxygen species (ROS) during acute shear stress (SS). We hypothesized that chronic SS attenuates the production of H2O2 during acute SS.Human microvascular endothelial cells (HMVEC) were exposed to one hour of laminar SS, either with or without a preceding 24 hour SS exposure (chronic SS). Cells were studied either in full serum media (5%) or after serum‐starving (to increase ROS) overnight (0.5% serum). 2′,7′‐dichlorofluorescein (DCF) assessed H2O2 formation with flow cytometry (N=3–7).Acute SS increased H2O2 release compare to static conditions (4.4±0.5 fold increase (F.I.) for 0.5% serum and 1.7±0.2 F.I for 5% serum (p<0.05)). Presence of L‐Nitro‐Arginine Methyl Ester (L‐NAME; nitric oxide synthase inhibitor) during acute shear reduced production of H2O2 in serum starved cells (2.7±0.2 F.I. vs. 4.4±0.5 F.I. (p<0.05)), but not in full serum. Acute SS after chronic SS reduced H2O2 in cells grown in low (1.9±0.8 F.I.) and full serum (0.9±0.1 F.I.). This reduction in H2O2 was reversed by L‐NAME in full serum (2.2±0.5 F.I. vs. 0.9±0.1 F.I. (p<0.05)), but not in low serum.Preconditioning with 24 hrs of SS inhibits generation of H2O2 during acute SS. We conclude that NOS modulates H2O2 production differentially in high and low serum conditions.
Saponin Selectively Permeabilizes the Sweat Duct Basolateral Membrane (BLM)
Authors: Reddy M.M. 
Published: June 2021

Understanding the role of endogenous phosphatases and kinases in regulating CFTR in the native epithelial cells is hindered by our inability to add or delete large, putative regulatory proteins to and from native cells at will. Therefore, we investigated the ability of Saponin to selectively permeabilize the BLM to macromolecules. We measured the CFTR Cl‐ conductance (CFTR‐gCl) in the sweat ducts as previously described (Quinton, 1986) and permeabilized the BLM either with α‐toxin (1000 U/ml) or with Saponin (0.01%) in the basolateral bath. ATP (5mM), cAMP (0.1 mM), PP2A inhibitor Okadaic acid (10−8 M) was added to the cytoplasmic bath as needed. α‐toxin permeabilization of the BLM spontaneously inhibited CFTR‐gCl (3.5±2 mS/cm2, n=12) Such deactivation was prevented either by the application Okadaic acid (CFTR‐gCl= 38± 4 mS/cm2, n=5) or cAMP CFTR‐gCl=45±6 mS/cm2, n=12). In contrast, after permeabilizing the BLM with saponin, CFTR‐gCl failed to deactivate if ATP was present. These results seem to indicate that: a.) α‐toxin pores in the BLM are small enough to retain endogenous phosphatases in the cytosol that dephosphorylate and deactivate CFTR even while depleting cytosolic cAMP through α‐toxin pores, b.) Saponin appears to make larger pores in the BLM so that endogenous phosphatases are lost from the cytosol that prevents deactivation by dephosphorylation of CFTR‐gCl.We thank Mr. K. Taylor for technical assistance. Supported by the NIH
Inhibition of Na+ transport in mouse lung exposed to chlorine (Cl2)
Authors: Lazrak Ahmed , Cain Laura , Doran Stephen , Matalon Sadis 
Published: June 2021

Lung fluid balance is finely tuned by vectorial transport of ions across the epithelium. Active sodium reabsorption generates the driving force needed for fluid clearance. In the inflammatory disease of the lung, high levels of Hypochlorus acid (HOCl) are generated by stimulated neutrophils. HOCl causes extensive damage through its reaction with cellular substances such as thiols and amines. In addition to the physiological sources, HOCl can be generated by inhalation of Cl2 known to induce lung edema. Herein; we investigated the effect of HOCl on ENaC in mouse lungs exposed to 400 ppm Cl2, one hour and 24 hours later using the single channel recording on live lung slices. The control alveolar cells exhibited two Na+ conductances, highly selective 4–6 pS and moderately selective 14–16 pS. Both were amiloride sensitive. One hour after exposure to Cl2, the Po of both channels was significantly reduced from 0.25 ± 0.05 to 0.06 ± 0.01 (p<0.001) and 0.12 ± 0.07 to 0.02 ± 0.005 (p<0.001) for highly and moderately selective channels, respectively. At 24 hours post exposure to Cl2 the Po of both channels recovered to 50% of its initial value in addition a new non selective conductance of 25 pS was observed. In experiments where animals were exposed to Cl2 and injected with a cocktail of antioxidants the recovery was faster, both pre‐existing channels recovered by 90% of their initial values. The non selective conductance (25 pS) was less active. Cl2 gas inhalation inhibits ENaC which may lead to alveolar edema formation. This injury was prevented by post administration of antioxidants.This Research was supported by NIH U01 ES015675 and U54 ES017218 grant to Sadis Matalon.
Regulation of Plasma Membrane Expression of TRPV4 Channel by GSK1016790A and Hypotonic Stress in HeLa and M‐1 Cells
Authors: Jin Min , James Jose , O'Neil Roger G. 
Published: June 2021

TRPV4 (Transient Receptor Potential Vanilloid 4) channels are activated by a wide range of stimuli, including hypotonic stress, phorbol ester 4α‐PDD, non‐noxious heat and mechanical stress. GSK1016790A (GSK101, GlaxoSmithKline), a potent and specific activator of TRPV4 was evaluated in HeLa cells transiently transfected with TRPV4 (HeLa‐TRPV4) and M‐1 (mouse cortical collecting duct) cells expressing TRPV4 endogenously. GSK101 (10 nM) causes a TRPV4 specific calcium influx in both over‐expressed and endogenous cell models for TRPV4, which can be inhibited by Ruthenium Red (RR). Hypotonic stress (225 mOsm/kg) causes a calcium influx in HeLa‐TRPV4 and M‐1 cells, but only partially responds to RR inhibition. Plasma membrane proteins were extracted and Western blotting was utilized to study the expression of TRPV4 over a time course of 30 minutes under GSK101 and hypotonic stress treatment. GSK101 causes a steady down‐regulation of TRPV4 plasma membrane expression over 30 minutes, while the hypotonic stress causes a more complex change in TRPV4 plasma membrane expression. Fluorescence confocal microscopy shows co‐localization of TRPV4 with microtubules and actin, suggesting likely trafficking and endocytosis of TRPV4 channel. Immunocytochemistry also shows co‐localization of TRPV4 with large‐conductance calcium‐activated potassium channels (BK), indicating a potential macro‐complex of these channels are probably involved in intracellular calcium and volume regulation. It is concluded that GSK101 specifically activates TRPV4 in HeLa‐TRPV4 cells and M‐1 cells, which likely involves regulation of endocytosis of TRPV4 from the plasma membrane. Hypotonic stress activates TRPV4 in a more complex pattern of regulation that may involve recycling.Supported by NIH DK70950.