bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating a 3‐step leukemogenic hypothesis in C57BL/6 mice: tungsten, RSV and hypoxia
Authors: Fastje Cynthiha D. , Harper Kevin , Terry Chad , Wong Simon S. , Witten Mark L.
Published: June 2021

Significantly elevated concentrations of tungsten have been demonstrated in the atmospheric particulate matter in multiple communities experiencing elevated rates of childhood leukemia, but not in their respective control communities. Prenatal exposure to sodium tungstate in C57BL/6 mice (2208mg/kg/day via aerosol and/or 0.0994mg/kg/day via drinking water) significantly decreased the transcriptome expression of Dmbt1, a protein which functions to aggregate bacteria and viruses in lung mucosa and saliva. Additionally, analysis of gene microarray data produced a significant network associated with hematological/immunological disease focused on genes involved in viral defense. We hypothesize that the etiology of environmentally‐induced leukemia is in utero exposure to metals such as tungsten (W) which may increase susceptibility to viral influences, post‐natal exposure to Respiratory Syncytial Virus (RSV), and triggered by a hypoxic event. End measures include qRT‐PCR of RSV genes F & G during the fastigium of the infection, RT2‐PCR for genes associated with immunological function, and a complete blood count with a differential. Mice in the W/RSV/Hypoxia group developed neutrophilia post‐hypoxic challenge and at five months developed lymphocytosis contributing to leukocytosis.

Supported by EPA Grant EM‐96963201
Repair and regeneration in a giant danio (danio aequipinnatus) model of heart Injury
Authors: Lafontant Pascal J , Burns Alan R , Grivas Jamie , Lesch Mary Ann , Frounfelter Tyler , Golden Benjamin L , Fitzharris Neil , Nida Berhanemeskel
Published: June 2021

We have studied the characteristics of the heart in the adult Giant danio (Danio aequipinnatus), the wound repair response and the ability to regenerate following cautery injury. Electron microscopy studies revealed cardiac myocyte ultrastructure and organization similar to that observed in the zebrafish (Danio rerio). Apical cauterization resulted in injury to 25% of the ventricle. The injury initiated a robust inflammatory response with early recruitment of heterophils and mast cells, and the persistence of myeloperoxidase‐positive cells beyond the first week post‐injury. Bromodeoxyuridine and proliferating cell nuclear antigen‐positive cells, including endothelial cells and cardiac myocytes, were present in areas adjacent to the site of injury during the repair process, but primarily in adjacent subepicardial compact heart region by the second week. We observed marked collagen accumulation on day 14 that persisted through day 45. The collagen accumulation did not result in permanent fibrosis but appears to be resolved by 60 days when the ventricle was completely regenerated. Our data suggest that the Giant danio possesses robust repair mechanisms in the ventricle and may serve as an important model of cardiac inflammation, remodeling and regeneration.
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
Mitochondrial p53/Bid interaction plays a pro‐apoptotic role in response to PPARγ and RXR ligands in breast cancer cells
Authors: Bonofiglio Daniela , Cione Erika , Vizza Donatella , Perri Mariarita , Pingitore Attilio , Qi Hongyan , Catalano Stefania , Panno Maria Luisa , Genchi Giuseppe , Andò Sebastiano
Published: June 2021

The combined treatment with nanomolar levels of PPARã ligand Rosiglitazone (BRL) and RXR ligand 9‐cis‐retinoic acid (9RA) induces a p53‐dependent apoptosis in MCF‐7, SKBR‐3 and T‐47D human breast cancer cells. In this study we demonstrated that PPARã and RXR ligands at low concentrations trigger apoptosis independently of p53 action at genomic level but related to its interaction with some members of Bcl‐2 family proteins in the mitochondria. For instance, the combined exposure to BRL and 9RA induced an upregulation of Bid expression in mitochondrial extracts of breast cancer cells tested. In the same circumstances, we observed an enhanced association between Bid and p53 in mitochondrial membrane immunoprecipitates. The modulatory role played by Bid in the apoptotic process induced by BRL and 9RA appears to be crucial since both the induction of PARP cleavage and the percentage of TUNEL‐positive cells were reversed after knocking down Bid. These data address how the combined low doses of PPARã and RXR ligands sustain a mitochondrial p53/Bid interaction in inducing breast cancer cells apoptosis.
Development and Characterization Of Cell Lines Derived From Hamster Breast Carcinomas As Model Of Oncolytic Virotherapy
Authors: Kleiner‐Hancock Heather E , Coburn Malari , Zhang Songlin , Cheng Bing , Li Linda , Lowery‐Nordberg Mary , Li Benjamin D.L. , Mathis James Michael
Published: June 2021

Two models of breast cancer to study oncolytic virotherapy were investigated in female Syrian golden hamsters. In contrast to most other species examined, the Syrian hamster is permissive for human Adenovirus replication. Single doses of 2 carcinogens to 2 different groups of animals were administered: 7,12‐dimethylbenz[a]anthracene (DMBA) or N‐methylnitrosourea (MNU). Body weights, palpations and PET imaging for tumors were recorded. Animals were necropsied and tissues were taken for primary culture, histology and H&E staining. After cell lines were established from primary tumors, karyotyping, infectivity, oncolytic activity, and growth rate were determined. Both lines contain 74 chromosomes where normal for hamster is 44. Histology determined the DMBA line was derived from a highly aggressive, poorly differentiated carcinoma. When injected into nude mice xenografts, the DMBA derived line grew rapidly, invaded into underlying muscle, and metastasized to lung and bone. The MNU line, clearly an adenocarcinoma, was syngeneicly transplanted into recipient hamsters with a take rate of <50%; those tumors were harvested, grown in primary culture, and transplanted again with a take rate of 100%. The MNU line shows promising results for use in oncolytic virotherapy while the DMBA might prove to be useful in testing for failed drugs. Funding: Feist‐Weiller Cancer Center and the LA Gene Therapy Consortium
Annexin A1 and cytostatic effects of HDAC inhibitors on breast adenocarcinoma cell line, MCF7
Authors: Hirata Aiko , Senanayake Thulani , Woster Patrick M , Hirata Fusao
Published: June 2021

Annexin A1 is a major substrate of oncogenic kinases such as c‐met and c‐src, and is thus proposed to be involved in signal transduction of growth factors and mitogens. Since the expression of annexin A1 is decreased in breast cancer and other cancers, this protein is thought to have suppressor functions in these cancers. In accord with this interpretation, its expression was increased in breast adenocarcinoma MCF 7 cells as treated with THS 79‐12, a polyaminobenzamide histone N‐deacetylase (HDAC) Class 1 inhibitor, but much less with the nonspecific HDAC inhibitor, SAHA. The epigenetic induction of annexin A1 paralleled the inhibition of HDAC 1 but not the degree of apoptosis and caspase 3 activation. The cell growth of HDAC inhibitors paralleled the degrees of annexin A1 induction, but not of p21. Annexin A1 apparently increased EGF activated p38 MAP kinase, STAT3 kinase and Erk kinase, but decreased Akt kinase. Similar, if not same, changes were also observed by transfection of annexin A1 cDNA into MCF7 cells. (Supported in parts by a grant from The Susan G. Komen Breast Cancer Foundation)
Sirtuin expression is downregulated in non‐small cell lung cancer
Authors: Xie Qian Reuben , Li Ziming , Chen Zhiwei , Lu Shun , Ying Weihai , Xia Weiliang
Published: June 2021

Sirtuins comprising a family of NAD+‐dependent protein deacetylases and ADP‐ribosyltransferases are emerging important proteins regulating metabolism, stress response, aging and cancer. The expression of SIRT1 has been reported in ovarian and colon cancers suggesting its diagnostic/prognostic significance. However, for other forms of sirtuins and other forms of cancers such knowledge is lacking. In this study, we examined the expression of sirtuins in non‐small cell lung cancer (squamous carcinoma and adenocarcinoma) patients. Paired tumor and normal lung tissues were obtained from patients (staging T2N1‐2M0, n=8, male, median age=52.5, randomly selected from a pool of 230 patients). Real‐time quantitative PCR and Western blot were performed to quantify the expression of sirtuins (SIRT1‐SIRT7). We found that SIRT1 (p<0.01), SIRT2 (p<0.01), SIRT4 (p<0.05), SIRT5 (p<0.05) were expressed significantly less in tumor than non‐tumor tissues. Our results suggest down‐regulation of sirtuins might be an important step for tumor progression in NSCLC, and other sirtuins including SIRT2, SIRT4 and SIRT5 together with SIRT1 could be considered for modulation to treat lung cancer. (Supported by “Rising Star” Grant from Science and Technology Commission of Shanghai and Young Investigator Grant from National Natural Science Foundation of China)
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]
Pharmacogenetic Testing in the Pediatric Epileptic Population
Authors: Sander Tara , Marcuccilli Charles J. , Zembles Tracy , Hines Ronald N. , Lo Stanley , North Paula E.
Published: June 2021

Epilepsy is a chronic neurologic disorder characterized by recurrent unprovoked seizures that can be controlled with antiepileptic drugs (AEDs) in approximately 65% of patients. However, AED therapeutic windows are narrow and patients can experience severe adverse drug reactions (ADRs). CYP2C9, CYP2C19, EPHX1, and ABCB1 genetic variants can affect the metabolism and transport of AEDs. A pharmacogenetic test of these loci was developed for use in first‐time seizure pediatric patients to guide therapy and ADR risk. Laboratory specimens and quality controls were analyzed using the ABI Taqman Drug Metabolism Genotyping Assays. Genotypes were detected on the ABI 7900HT Fast Real‐Time PCR system. Testing was validated using human genomic specimens, customized plasmid DNA, and oligonucleotides representing all tested alleles. The assays demonstrated 100% specificity and 100% accuracy in detecting the correct genotype for synthetic DNA and 48 human genomic samples. To date, 53 patients have been tested. Genotype results were in agreement with expected allelic frequencies in the tested population. Test results were evaluated by a Pharmacogenetics resource team to individualize AED therapy. For 10% of these patients, the results were beneficial and provided faster therapeutic treatment. For some patients, knowing the reference genotype was useful in providing greater physician comfort of the prescribed AED.
Egr3 is a transcriptional regulator required for normal target tissue innervation during sympathetic nervous system development
Authors: Quach David Hon , Li Lin , Eldredge Laurie , Honasoge Avinash , Gruner Katherine , Tourtellotte Warren
Published: June 2021

Nerve Growth Factor (NGF) is a target tissue‐derived neurotrophin that is essential for normal sympathetic nervous system (SNS) development. Egr3 is regulated by NGF signaling and in Egr3‐deficient mice sympathetic neuron loss and target tissue innervation abnormalities lead to physiologic dysautonomia. It has not been clear to what extent Egr3 regulates sympathetic neuron survival or target tissue innervation. Here, we show that Egr3 has no effect on NGF‐dependent neuron survival or neurite outgrowth in vitro, but that inhibition of sympathetic neuron death in the absence of Egr3 in vivo leads to target tissue innervation abnormalities. To better understand the mechanism of Egr3 action, microarray analysis was used to identify potential target genes regulated by Egr3 in developing sympathetic neurons. We identified two potentially interesting genes, Hs3st2 and Esd which were confirmed to be downregulated in the absence of Egr3 and the promoters were confirmed to be transactivated by Egr3. Hs3st2 and Esd may be involved in the modification of carbohydrate molecules located on cell surface membranes, and several studies indicate that carbohydrate moieties can be essential for normal axon guidance and branching. Thus, Egr3 may regulate some target genes involved in the modification of cell surface molecules that have a role in sympathetic terminal axon invasion and ramification within target tissues.
Phosphatidylcholine supplementation alleviates the impaired glucose response induced by acute methionine loading
Authors: Lopez Anna‐lee N , Gossell‐Williams Maxine D , Simon Oswald R , Phillips Howard
Published: June 2021

Methionine metabolism liberates homocysteine. Emerging evidence suggests acute hyperhomocysteinemia may play a critical role in impaired insulin secretion from pancreatic beta cells and thus could prevent the normal response to a glucose challenge. The aim of the study was to evaluate the benefits of supplemental phosphatidylcholine (PC) on the glucose response associated with an oral glucose tolerance test (OGTT) in rats subjected to acute methionine loading. Male Sprague‐Dawley rats were divided into groups for corn oil (n=4) and PC supplementation, 458mg/kg (n=4), by gavage 5 days weekly for 3 weeks. Animals were fed a standard pellet diet and tap water ad libitum. OGTT and methionine loading was conducted after supplementation. Fasting blood glucose concentration was unaffected by treatment with PC. Impaired glucose tolerance was observed in control group (7.48 ± 0.14) at 2hrs post methionine and glucose load. Conversely, PC supplementation resulted in a significant reduction in glucose levels at 1.5hrs and 2hrs when compared to controls (P<0.05 and P<0.001 respectively). Additionally, values approaching significance were also recorded at 1hr (P=0.055). PC supplementation may be very effective in preventing the impaired glucose tolerance associated with decreased insulin secretion that can occur with acute hyperhomocysteinemia. Source of funding: Office of Graduate Studies and Research, UWI Mona
Effect of Pioglitazone, Simvastatin and Their Combination on Adiponectin and TNF‐α Level in the Metabolic Syndrome: Novel Findings from the High Fructose‐Fed Rat Model.
Authors: Nassar Noha N , Schaalan Mona F
Published: June 2021

Hypoadiponectinemia and increased tumor necrosis factor‐α (TNF‐α) play an important role in the development of insulin resistance (IR), The current study aimed to investigate the effect of pioglitazone (PIO) and simvastatin (SIM) on serum levels of adiponectin and (TNF‐α), as well as on the metabolic perturbations and disturbed homeostasis of glucose and lipids in a fructose‐induced insulin resistance (HFD) rat model. To this end sixty male Wistar rats were divided into two dietary regimen‐groups for a period of 8 weeks. Ten rats were fed normal chow (control). Fifty rats were fed a fructose rich chow (60% fructose, 5% fat and 20% protein) for 8 weeks. Following confirmation of IR, rats the latter group were further subdivided into 4 groups (10 rats; each). GpI: high fructose diet group (HFD); Gp II: HFD + PIO (10 mg kg−1); GpIII: HFD + SIM (20 mg kg−1); Gp IV: HFD + a combination of PIO/SIM for two weeks. Fructose‐fed rats developed (IR) manifested as increase in body weight, glucose levels, HbA1c HOMA‐IR index, TGs, T‐Chol, LDL‐C and significant decrease in HDL‐C. Hypoadiponectenemia, increased serum TNF‐α and ratios of epidedymal fat/body weight, visceral fat/body weight and liver weight/body weight were prominent findings in this study. Moreover, positive correlation between HOMA IR‐index and TNF‐α and a negative one with adiponectin were revealed. Treatment with PIO or SIM significantly reduced metabolic parameters as well as a decrease in TNF‐α and increase in adiponectin serum level were observed, Co‐administration of SIM and PIO synergistically enhanced the overall metabolic parameters.
Potential Utility of Single and Concomitant Administration of Pioglitazone and Simvastatin In Ameliorating Endothelial Dysfunction In Diabetic Insulin Resistant Rats.
Authors: Nassar Noha N , Schaalan Mona F
Published: June 2021

Reported studies link insulin resistance (IR) to the development of endothelial dysfunction, a hallmark of cardiovascular diseases. Features of endothelial dysfunction correlate increased inflammatory mediators. In an attempt to ameliorate the endothelial perturbations and concomitant inflammatory process induced in IR, Pioglitazone (Pio) and Simvastatin (Sim) and their combination were used to treat fructose fed insulin resistant (FFIR) rats. To this end, Sixty Wistar rats were assigned to 6 groups (10 rats, each) as follows; GpI: control receiving standard chow for 8 weeks,; GP II and GP III (FFIR) or FFIR + vehicle and sacrificed afterwards; GP IV FFIR + PIO (10 mg/kg,p.o); GP V FFIR+ SIM (20mg/kg,p.o) and GP VI: FFIR+ PIO/SIM for the last two weeks of the total 8 weeks. The FFIR rats manifested with significant hyperglycemia, hyperinsulinemia, dyslipidemia, hypoadiponectenemia and high TNF‐α level associated with significant increases of AST, LDH, CPK, CKMB and troponin. Moreover investigation of endothelial function revealed significant increases in homocysteine, VCAM, hsCRP and VEGF. Both Pio and Sim, albeit Sim to a greater extent, ameliorated the assessed parameters. Moreover the combination proved better than each alone. The biochemically measured parameters were further confirmed with histopathological examination of aortas from the treated vs FED animals. In conclusion, we demonstrate a link inflammatory metabolic processes with cardiovascular events and the reversal of these unwanted events by administration of both drugs which exceeded the beneficial effect of each alone in modulating endothelial function
Changes in angiotensin II type 2 (AT‐2) receptor function in the aorta of diabetic rats over time
Authors: Sabra Ramzi , Al‐Hariri Ahmad , Ghali‐Ghoul Rana
Published: June 2021

We investigated the response to angiotensin II (AII) and CGP42112 (an AT‐2 selective agonist) in the presence of losartan, in aortic rings from control rats and rats made diabetic by streptozotocin (85 mg/kg i.v.), 2 and 12 weeks after injection. Responses were obtained in intact and endothelium denuded aortic rings pre‐contracted with 1 μM phenylephrine (PE). At 2 weeks of diabetes, AII induced no response in intact rings from control rats, whereas rings from diabetic rats were relaxed up to a maximum of 80% of the PE‐induced contraction. In the absence of endothelium, the relaxant response was annulled. CGP42112 also induced greater vasodilation in diabetic rings, but the relaxation was more prominent in the rings devoid of endothelium. 12 weeks after streptozotocin, the response to AII in endothelium‐intact rings was reversed, whereby rings from diabetic rats had a lower degree of relaxation than controls, with the latter showing significant relaxation, in contrast to the situation at 2 weeks. Removal of endothelium annulled the relaxation in both rings. The response of intact rings to CGP42112 was also less in rings from diabetic rats compared with controls, but removal of endothelium rendered the relaxation more prominent in rings from diabetic rats, similar to what was observed at 2 weeks. These results suggest that the activity of AT‐2 receptors varies over time in diabetic rats, and that the responses to AII and CGP42112 in the presence of losartan are not identical, with endothelium derived vasodilators playing a greater role in the response of the former.
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.

Conclusion

ClC‐3 may be responsible for the constitutively‐activated VRCCs in hypertrophied heart. (supported by NCRR P‐20 RR‐15581)
Monoamine oxidase B gene deletion prevents cardiac pump dysfunction in mice with pressure overload
Authors: Kaludercic Nina , Takimoto Eiki , Nagayama Takahiro , Lai Edwin , Chen Kevin , Shih Jean C , Pacak Karel , Kass David A , Di Lisa Fabio , Paolocci Nazareno
Published: June 2021

Monoamine oxidase A (MAO‐A) inhibition prevents pressure overload‐induced congestive heart failure (CHF), decreasing oxidative stress due to MAO‐catalyzed degradation of norepinephrine (NE) and rescuing sympathetic NE content (Kaludercic et al., Circ. Res., 2009). MAO‐B is the major isoform present in the mouse myocardium responsible for catabolism of both NE and dopamine (DA), both endowed with cardiostimulatory effects. Yet whether it contributes to CHF unfolding is unclear. WT and MAO‐B knockout mice (MAO‐B−/−) were subjected to CHF via transverse aortic constriction (TAC, 9 weeks). LV function was assessed by pressure‐volume (PV) analysis. After TAC, WT hearts had increased left ventricle (LV) dimensions and impaired function accompanied by markedly lower levels of tyrosine hydroxylase, the rate limiting enzyme in DA biosynthesis. Conversely, MAO‐B−/− mice showed preserved end‐systolic (ESV) and end‐diastolic (EDV) volumes and improved LV function: ESV was 5.5±0.8 in MAO‐B−/− vs 16.6±4.5 μl in WT and ejection fraction was 79±1 vs 59±8% in WT (both p<0.01). MAO‐B−/− mice had increased basal DA content. DA (1mM) directly applied to WT isolated cardiac myocytes markedly increased sarcomere shortening (+78%, p<0.05), improving relaxation (−15%, p<0.05). In conclusion, MAO‐B activity shapes basal cardiac function and when inhibited prevents LV remodeling, thus sustaining inotropy in failing hearts.
8‐pCPT‐Conjugated Cyclic AMP Analogs exert Thromboxane Receptor Antagonistic Properties
Authors: Schmidt Martina , Sand Carsten , Grandoch Maria , Weernink Paschal Oude
Published: June 2021

Membrane‐permeable 8‐pCPT‐2′‐O‐Me‐cAMP has been shown to specifically activate cAMP‐regulated Epac proteins, without direct effects on protein kinase A and protein kinase G. During isometric tension measurements in thoracic aortic rings from Wistar rats, we observed that 8‐pCPT‐2′‐O‐Me‐cAMP selectively induced a rightward shift of the concentration response curve for the thromboxane mimetic U46619, without altering the contractile response to noradrenaline. We hypothesized that 8‐pCPT‐2′‐O‐Me‐cAMP and similar compounds may function as direct thromboxane receptor antagonists. Indeed, in addition to 8‐pCPT‐2′‐O‐Me‐cAMP, also 8‐pCPT‐cAMP, Rp‐8‐CPT‐cAMPS and 8‐CPT‐adenosine, but not 8‐Bromo‐2′‐O‐Me‐cAMP, induced rightward shifts of the contractile response to U46619. Likewise, 8‐pCPT‐2′‐O‐Me‐cAMP and Rp‐8‐CPT‐cAMPS, but not 8‐Bromo‐2′‐O‐Me‐cAMP, specifically reduced U46619‐induced aggregation of human platelets. In addition, 8‐pCPT‐2′‐O‐Me‐cAMP and Rp‐8‐CPT‐cAMPS completely reversed U46619‐induced reduction of intercellular adhesion molecule‐1 expression and migration of human coronary artery endothelial cells. Most important, the cAMP analogs that reduced the contractile response to U46619 also concentration‐dependently inhibited binding of the thromboxane receptor radioligand [5,6‐3H]SQ29548 to human platelets. We conclude that 8‐pCPT‐conjugated cAMP analogs exert competitive thromboxane receptor antagonistic properties. Supported by the Deutsche Forschungsgemeinschaft and a Rosalind Franklin Fellowship from the University of Groningen.
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

Objective

Pentaerithrityl 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/Results

Recombinant 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.

Conclusions

This study revealed that PETN has intrinsic antioxidative properties and does not trigger ALDH‐2‐dependent ROS formation from the purified enzyme.
Enhancement of the Rostral Ventrolateral Medulla ERK1/2/nNOS/NO Signaling Pathway is Implicated in the Central Cannabinoid Receptor‐Evoked Sympathoexcitation in Conscious Rats
Authors: Ibrahim Badr Mostafa , Abdel‐Rahman Abdel A.
Published: June 2021

Reported studies have shown that activation of CB1R in the rostral ventrolateral medulla (RVLM) of anaesthetized animals results in sympathoexcitation/pressor response; yet, the exact signaling mechanisms are unknown. Moreover, it has been shown that phosphorylation of molecular targets ERK 1/2 and nNOS (p‐ERK1/2 and p‐nNOS), which led to NO release underlies wide array of CB1R‐evoked pharmacological actions. Therefore, this study aimed to elucidate the role of p‐ERK1/2‐p‐nNOS‐NO signaling in the central CB1R‐mediated sympathoexcitation. We have simultaneously measured RVLM‐NO levels in real‐time (in vivo electrochemistry), blood pressure and heart rate following unilateral intra‐RVLM microinjection of CB1R agonist [(R)‐(+)‐[2,3‐Dihydro‐5‐methyl‐ 3[(4‐morpholinyl)methyl]pyrrolo[1,2,3‐de]‐1,4‐benzoxazinyl]‐(1‐naphthalenyl) methanone mesylate salt] WIN55212‐2 (100, 200 or 300 pmol/80nL, n=5) in conscious rats. WIN55212‐2 elicited dose dependent increases in blood pressure and RVLM‐NO levels (n=5, P<0.05). The vehicle of WIN55212‐2 had no effect on the measured variables. Additionally, ERK1/2 and nNOS phosphorylation was significantly higher in the injected RVLM compared to control (n=4, P < 0.05). These findings provide new insight into molecular mechanisms that underlie the central CB1R‐evoked pressor effects.
CART peptide may be an endogenous antidepressant
Authors: Job Martin , McNamera Ilan , Kuhar Michael
Published: June 2021

CART p (cocaine and amphetamine regulated transcript peptide) may be an endogenous antidepressant. Anatomically, CART mRNA is situated in various brain regions that are thought to be involved in depression, and CARTp interacts with various neurochemical systems associated with mood, stress and reward. In a human study (Miraglia del Giudice et al., 2006, Depress Anxiety 23:90–92), it was discovered that subjects carrying a missense mutation of the CART gene displayed an increase in depression. Also, in animal studies, injection of CARTp reduced immobility time in the forced swim test (Dandekar et al., 2009, Neuropsychopharmacology 34:1288–1300). Interestingly, electroconvulsive therapy, which is an effective treatment for depression in humans, increased CART mRNA expression in the nucleus accumbens of rats. Furthermore, CARTp upregulates BDNF (brain derived neurotrophic factor ‐ which is thought to play an essential role in the mechanism of antidepressants) levels in hippocampal neurons. The conclusion from all these studies is that CART may play an important role in the etiology of depression and in the mechanism of antidepressants, and may likely be an endogenous antidepressant. Source of research support ‐ RR00165, DA15162, DA015040.
Trace amine associated receptor 1 mediates methamphetamine effects on monoamine transporter function
Authors: Miller Gregory M , Xie Zhihua
Published: June 2021

Trace amine associated receptor 1 (TAAR1) is activated by a spectrum of biogenic amines, amphetamine‐like compounds and thyronamines. It is present in brain monoaminergic regions and co‐localized with the dopamine transporter (DAT) in some neurons. Here, we determined whether TAAR1 signaling in response to methamphetamine (METH) is involved in non‐competitive effects of METH on DAT, norepinephrine transporter (NET) and serotonin transporter (SERT) regulation. Uptake and efflux assays were performed in monoamine transporter/TAAR1 co‐transfected cell lines and in rhesus monkey, wild‐type (WT) and TAAR1 knockout mouse (KO) striatal ([3H]DA) and [3H]5‐HT) and thalamic ([3H]NE) synaptosomes. METH activation of TAAR1 resulted in enhancement of uptake inhibition and promotion of efflux of [3H]monoamine by DAT, NET or SERT in vitro and ex vivo, across high and low loading concentrations of [3H]monoamine. At low loading concentrations (10–20 nM [3H]monoamine), METH action on uptake and efflux was entirely TAAR1‐ and PKA‐/PKC‐dependent. Further experiments revealed that 1 μM METH‐induced DAT internalization was TAAR1‐ and PKC‐dependent in vitro and ex vivo. Accordingly, METH targets and interacts with TAAR1 to initiate cellular phosphorylation cascades that regulate monoamine transporter function and trafficking. Support: NIH DA022323, DA016606, DA025697, DA025802 and RR00168
Visualizing RNA interference mediated knockdown of the 5‐HT1A receptor
Authors: Dudek Nichole L , Vantrease Jaime E. , Scrogin Karie E.
Published: June 2021

Methods were developed to optimize testing of small hairpin RNA (shRNA) sequences targeting the rat 5‐HT1A receptor (5‐HT1AR). Five anti‐5‐HT1AR shRNA sequences were cloned into a vector containing a U6 promoter to drive shRNA expression and a CMV promoter to drive green fluorescent protein expression. A construct encoding the 5‐HT1AR fused to mCherry fluorescent protein (5HT1AR‐cherry) was also developed. To screen shRNAs, HEK‐293 cells were transfected with 5HT1AR‐cherry and a vector containing an anti‐5‐HT1AR shRNA sequence, or a control vector encoding a shRNA targeted to rat tryptophan hydroxylase 2 (TPH2‐shRNA). One shRNA, targeting mRNA coding for a portion of the third intracellular loop of the 5‐HT1AR, termed 1AR(v2)‐shRNA, was found to produce effective knockdown of the 5‐HT1AR as evidenced by decreased mCherry fluorescence. Cells expressing 5HT1AR‐cherry and 1AR(v2)‐shRNA had decreased 5‐HT1AR mRNA levels (1.6 fold decrease, P<0.05) compared to cells expressing 5HT1AR‐cherry and TPH2‐shRNA. Cells expressing an untagged form of the 5‐HT1AR and 1AR(v2)‐shRNA had reduced 5‐HT1AR protein levels compared to cells expressing 5‐HT1AR and TPH2‐shRNA (4.81±0.61 vs. 0.33±0.04 NU, P<0.005). Future studies will utilize an adeno‐associated virus to mediate expression of 1AR(v2)‐shRNA to assess the role of 5‐HT1A receptors in the sympathetic compensatory responses to hemorrhage.
Desipramine is a novel modulator of norepinephrine‐induced signaling through the alpha2A adrenergic receptor
Authors: Cottingham Christopher Michael , Wang Qin
Published: June 2021

The objective of the current study was to characterize the antidepressant drug desipramine (DMI) as a direct ligand of the α2A adrenergic receptor (AR) and its ability to induce receptor responses such as trafficking and signaling. DMI was found to bind directly to the receptor via radioligand binding assay, consistent with previous reports. DMI is able to drive receptor trafficking (internalization measured by ELISA and quantitative immunofluorescence) in mouse embryonic fibroblasts (MEFs) and primarily cultured neurons. DMI alone is silent with regard to signaling, measured either by G protein activation or downstream effector (ERK1/2, Akt) activation. However, we have observed that DMI is able to influence signaling induced by the endogenous adrenergic ligand norepinephrine (NE): DMI, when given in combination with NE, is able to significantly attenuate the NE‐induced activation of Akt while causing a modest increase in NE‐induced activation of ERK1/2. These results indicate that this modulation by DMI has differential effects on downstream targets, rather than a general potentiation or attenuation of NE‐induced signaling. Regulation of the α2AAR by DMI may have implications for the in vivo antidepressant effects of this drug, although further studies will be necessary to investigate this. Work supported by NIH grant MH081917 (QW) and the Alabama State Fund through UAB.
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.
Interactions between the serotonin/norepinephrine uptake inhibitor clomipramine and mu opioid agonists in rhesus monkeys: role of mu agonist efficacy
Authors: Banks Matthew L , Negus Sidney S.
Published: June 2021

Adjunct medications may increase the efficacy or safety of μ opioid agonists for the treatment of pain. This study examined interactions between the serotonin/norepinephrine uptake inhibitor clomipramine and different efficacy μ‐opioid agonists (nalbuphine<morphine<fentanyl<methadone) in assays of thermal nociception (50 and 54°C warm water tail withdrawal) and schedule‐controlled responding (fixed‐ratio 30 schedule of food delivery) in rhesus monkeys. In the assay of thermal nociception, clomipramine was ineffective, and μ agonists produced efficacy‐dependent antinociception. Clomipramine produced superadditive effects in combination with the lower efficacy μ agonists nalbuphine and morphine, but only additive effects in combination with the higher‐efficacy μ agonists fentanyl and methadone. In the assay of schedule‐controlled responding, clomipramine was ineffective, and all μ agonists decreased response rates. Clomipramine produced additive effects in combination with all mu agonists. Clomipramine selectively enhanced the antinociceptive effects relative to the rate‐decreasing effects only for lower efficacy μ agonists. Research supported by NIH grants RO1‐DA11460 and T32‐DA007027.
NSAIDs Inhibit Activity and Reduce Surface Expression of Calpain Proteases
Authors: Silver Kristopher , LeLoup Ludovic , Freeman Lisa C , Wells Alan , Lillich James D
Published: June 2021

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are among the most commonly used drugs worldwide; however, chronic treatment with NSAIDs can result in gastrointestinal toxicity (GI). Previously, we have reported that NSAIDs associated with GI damage inhibit migration of cultured epithelial cells and suppress the expression of calpains, a family of cysteine proteases important in many cellular processes including cell migration. Accordingly, we examined calpain activity in monolayers of intestinal epithelial cells treated with NSAIDs using a calpain‐selective fluorescent reporter in living cells (t‐BOC). Additionally, we used a cell membrane footprinting technique to examine expression of calpains at the cell membrane attached to the substrate. Our results demonstrate that NSAIDs with ulcerogenic potential depress calpain activity within 24 h of treatment. Furthermore, NSAID treatment also inhibited localization of calpain proteases to the basal cell membrane and disrupted the formation of actin filaments. Thus, inhibition of epithelial cell migration by NSAIDs may result from disruption of calpain signaling and altered cytoskeletal organization. As these represent activities that are in addition to and not responsible for the immunosuppressive actvity, these findings open avenues for adjuvant treatments to minimize these morbid and even mortal consequences of these toxicities.
Bisphosphonate targeting to specific skeletal sites in mice with whole body vibration
Authors: Papineni Rao V , Orton Sean , Ji Tao , Schmitthenner Hans , McLaughlin William , Vizard Douglas , Pan Jingyi
Published: June 2021

The prospects of a combined targeting, and monitoring of the drug binding to specific skeletal sites by noninvasive means is determined. Site‐selective skeletal targeting of bisphosphonates, if feasible, is ideal to overcome drug‐related side effects. We utilized near‐infrared (NIR) fluorescence imaging to monitor the noninvasive delivery of NIR dye conjugate bisphosphonate, and the role of whole body vibration (WBV) to aid drug targeting. Vibration loading, utilizing whole body vibration (WBV) was performed on twelve mice by subjecting them to vibration frequency of 35 Hz for fifteen min (5–10 times/week) duration. Alendronate was conjugated to near‐infrared tricarbocyanine, cyclic enamine‐functionalized dye, and was injected intravenously after 21‐day exercise regime; the mice were imaged using a commercially available imaging system, KODAK in‐Vivo Multispectral FX system. Besides several specific targeting sites, NIR‐alendronate probe binding to the lumbar L6 vertebrae was explicitly observed, resultant of direct high‐frequency WBV stimulation. The targeted drug binding in response to the vibratory protocol provides hope in the development of whole body vibration (WBV) as a bisphosphonate‐therapeutic aid, and to develop strategies in reducing the drug dosage in osteoporosis treatment.
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.
The interaction of G protein‐coupled receptor kinase 4γ with Gαs is required for inhibition of the β2‐AR
Authors: Andresen Bradley T. , Flynn Megan K. , Sisk Kay , Bastepe Murat , Jackson Edwin K.
Published: June 2021

Polymorphisms in G protein receptor kinase 4γ (GRK4γ) have been implicated in hypertension via desensitizing Gs‐coupled receptors, such as the β2‐adrenergic receptor (β2‐AR). However, the molecular mechanism initiating phosphorylation and desensitization is unknown. Previous data demonstrate that GRK4γ interacts with inactive Gαs; thus, we hypothesized that the interaction of GRK4γ with Gαs is required for proper desensitization of the β2‐AR. To test this, we identified Gαs constructs that do not fully interact with GRK4γ and used these constructs in functional studies utilizing GnasE2‐/E2‐ mouse embryonic fibroblasts (MEFs), which are functionally Gαs knockouts. The data suggest that GRK4γ binds to Gαs; however, the precise area and amino acids involved in binding has yet to be identified. Moreover, the data confirm that expression of Gαs in GnasE2‐/E2‐ MEFs restores isoproterenol‐mediated generation of cAMP, which is inhibited by GRK4γ; however, when GRK4γ does not bind to Gαs it cannot inhibit β2‐ARs. In conclusion, the data suggests that GRK4γ binds to inactive Gαs positioning it near the receptor, and upon activation of the receptor Gαs dissociates from the receptor, Gβγ, and GRK4γ, resulting in signaling; then GRK4γ, which has previously been reported to be constitutively active, has access to the receptor and phosphorylates and desensitizes the receptor. This work was supported by the AHAF and is supported by a VISN 15 grant.