bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Soy Pinitol on Hepatic Lipid and Antioxidant Metabolism in High Fat Diet‐Fed C57BL/6 Mice
Authors: Choi Myung‐Sook , Kim Hye‐Jin , Do Gyeong‐Min , Park Hae‐Jin , Shin Su‐Kyoung , Choi Ji‐Young , Jeon Jong‐Eun , Jeon Seon‐Min
Published: June 2021

To investigate the body fat‐lowering, hypolipidemic and antioxidant effects of pinitol supplementation, we carried out to the animal experiment using mice fed high‐fat diet. In these results, 0.05% and 0.1% supplemenation of pinitol significantly suppressed the hepatic triglyceride and cholesterol accumulation, inhibited the hepatic cholesterol biosynthesis and conversion of storage form of cholesterol, and decreased hepatic fatty acid and/or triglyceride synthesis. And also antioxidant metabolism and hepatocytotoxicity were significantly improved by pinitol supplementation. Pinitol, however, did not affect the reduction of body weight or body fat weights, did not improve the plasma lipid profiles and insulin resistance. Accordingly, it is considered that soy pinitol supplementation is not significantly effective to the reduction of body weight and/or body fat, however, it is effective to the protection of hepatocyte and improvement of antioxidant metabolism compared to the HF group. In conclusion, the fucoxathin supplementation with a high‐fat diet seemed to be effective for suppressing body weight gain and for improving plasma and hepatic lipid metabolism in mice.
Experimental in vitro arterial reactivity and tissue culture solutions alter the time dependent stability of anthocyanins from chokeberry, bilberry and elderberry extracts.
Authors: Bell David Robert , Nalliah Ruth Ellen , Phillips Jordan S , Gaier Abby J , Jones Francis L , Gochenaur Kristen E
Published: June 2021

Acute exposure to anthocyanin enriched extracts from chokeberry (Ch), bilberry (B) and elderberry (E) produces endothelium‐dependent, NO mediated vasorelaxation of porcine coronary arteries. Extracts at 0.05mgTa/L also attenuate impaired vasorelaxation resulting from external or internal oxidant stress. Determining the stability of these extracts in in vitro settings is necessary to evaluate their long term effects on arteries. Therefore, we examined the stability of monomeric anthocyanins from Ch, B and E in common in vitro arterial function solutions. Reference extract solutions (0.75mg/ml in DD H2O at 21C)showed less than 25% monomeric anthocyanin degradation over a period of 9 days as determined by the pH differential method. Solutions made with a physiological salt solution (PSS) used for in vitro arterial reactivity studies showed significantly greater degradation at 21C.(1/2 life, hrs: Ch= 13, E=35, B=4.6). Degradation in PSS was increased 3–4 fold at 37C and 2 fold more with 95%O2/5%CO2 added. Similar results were seen in Dulbecco's modified Eagle's media solutions at 37C. Analysis of degradation by the bisulfite method revealed time dependent anthocyanin polymerization in in vitro arterial solutions. Thus solutions used to study arterial function in vitro produce significant degradation and polymerization of monomeric anthocyanins from Ch, B and E. These factors must be taken into account in the design and interpretation of studies that examine long term effects of anthocyanins on arterial function in vitro. (Supported by IUSM REGrant #39407‐1090 and the Lutheran Foundation of Indiana Grant # 6721712)
Piceatannol induces apoptosis through death receptor and mitochondrion‐dependent pathways in human prostate cancer cells
Authors: Park Heesook , Kim Eun Ji , Park So Young , Kwon Dae Young , Park Jung Han Yoon
Published: June 2021

Piceatannol (3,3′,4,5′‐tetrahydroxy‐trans‐stilbene) is a polyphenol found in grapes, red wine, and Rheum undulatum. Previous studies by others showed that piceatannol inhibits proliferation of various cancer cells. In this study we examined the effects of piceatannol on the growth of androgen‐sensitive LNCaP and androgen‐insensitive DU145 prostate cancer cells. Piceatannol decreased the viable numbers of DU145 and LNCaP cells and increased apoptotic cell numbers in a dose‐dependent manner. Western blot analysis revealed that piceatannol increased the protein levels of cleaved caspase‐8, ‐9, ‐7 and ‐3 and cleaved poly (ADP‐ribose) polymerase (PARP). Piceatannol increased mitochondrial membrane permeability and release of cytochrome c from mitochondria to cytosol. Piceatannol increased the levels of truncated‐Bid, Bax, Bik, Bok and Fas but decreased the levels of Mcl‐1 and Bcl‐xL. Caspase‐8 and caspase‐9 inhibitors mitigated piceatannol‐induced apoptosis. The caspase‐8 inhibitor suppressed piceatannol‐induced cleavage of Bid, caspase‐9, caspase‐3 and PARP. These results suggest that piceatannol induces apoptosis through the death receptor and mitochondrial dependent pathways.
Policosanol fails to lower HMG‐CoA reductase activity in HepG2 cells
Authors: Guderian David M. , Lee Ji‐Young , Carr Timothy P.
Published: June 2021

The cholesterol‐lowering properties of policosanol are controversial. One research group in Cuba have reported that policosanol intake of 5–10 mg/day in humans can lower plasma LDL cholesterol up to 25%, although other laboratories have failed to repeat these findings. Moreover, the cellular/molecular effects of policosanol are uncertain. Using HepG2 cells, we assessed the regulation of HMG‐CoA reductase (HMGR) activity and gene expression by incubating cells for 1.5, 3.0 or 4.5 hrs with bile salt micelles (taurocholate, lecithin) with and without policosanol. Policosanol uptake into HepG2 cells was confirmed by gas chromatography, demonstrating the efficacy of our micelle delivery system. Our results indicated no significant differences at any time point between control and policosanol treated cells; however, after 4.5 hrs of incubation, HMGR activity was significantly increased in both control and policosanol treated cells, suggesting that prolonged exposure to micelles may promote cholesterol depletion in HepG2 cells. We also quantified mRNA gene expression of HMGR and its transcription factor SREBP2. HMGR mRNA abundance was determined by real‐time PCR and indicated no difference in gene expression after 3 hrs. We conclude that policosanol has little or no effect on HMGR activity or gene expression in HepG2 cells. [Supported by USDA‐NRI Competitive Grant No. 2004‐35503‐14824]
ORAL FEEDING OF A MIXTURE OF FLAVONOIDS MODIFIES TISSUE COPPER AND ZINC CONCENTRATIONS IN RATS.
Authors: Bebe Frederick N , Panemangalore Myna
Published: June 2021

High level consumption of flavonoids as dietary supplements could increase the risk of metabolic alterations and perhaps toxicity. The objective was to determine the effect of administering different levels of a purified flavonoid mixture on changes in the concentration of zinc and copper in tissues. Male SD rats (150–175g, 6/group) had free access to the AIN‐93 diet and distilled water. A mixture of equal amounts of flavonoids (quercetin, rutin and catechin) at 5, 25, 50, 100 and 200 mg/kg bw (FM5, FM25, FM50, FM100, FM200) was dissolved in PEG and fed by gavage (0.5 mL/day/5days a wk; 0.5 mL PEG/CON) for 30 days. Plasma Cu decreased by about 25%–30% in all FM groups without a concomitant decrease in ceruloplasmin activity compared to CON. Liver: Cu declined by about 20% in all FM groups. Kidney: Cu declined by 20% (FL50 and FL100) and by 44% in FL200, while Zn increased by 20%, 24% and 34% in FL50, FL100 and FL200 compared to CON (P<0.05). Cu retention declined by 35% and 58% in the small intestines (SI) and by 17% and 26% in SI mucosa of FL100 and FL200 groups compared to CON, respectively (P<0.05). These changes in tissue Cu and Zn concentrations together with our previous data suggest that consumption of high levels of purified flavonoids could compromise Cu and to some extent Zn homeostasis in tissues. This is critical as the intake of Cu is largely marginal in most US population groups. (USDA#KYX‐50‐03‐17H).
Secretion of intestinal hormones is regulated by the consumption of nopal
Authors: Lopez Patricia , Ordaz Guillermo , Tovar Armando R , Torres Nimbe
Published: June 2021

The nopal (Opuntia sp), a Mexican variety of cactus is widely used as a food and as a popular treatment for diabetes mellitus. It has been seen that consuming nopal improves glucose tolerance. To determine whether nopal has an effect on insulin secretion, several gut hormones were measured after the consumption of 93.75 g of nopal beverage containing 50 g of available carbohydrates compared with a solution containing 50 g of glucose in a two hour period. Our results showed that the glycemic and insulinemic index of nopal were 32.1 + 10.7 and 7.8 + 1.2 respectively. The release of amylin, and GLP‐1 were lower in subjects that drank the nopal beverage. Interestingly GIP release was inhibited by 74 % in subjects that ingested the nopal beverage. Ghrelin was increased during the fasting and decreased more slowly after the consumption of nopal. The PP peptide increased significantly after 15 min of nopal consumption and decreased slower than the glucose solution. There was no significant difference in the PYY peptide with time. The results suggest that nopal modifies the release of several intestinal peptides decreasing insulin secretion and possibly alters hunger behaviour. Supported by: Nutriva de México S.A. de C. V. Aguascalientes, Ags. México.
Anti‐Inflammatory Activity of Fermented Fig Vinegar
Authors: Jeong Mi‐Ran , Jang Yong‐Suk , Yun Soon‐Il , Lee Young‐Eun
Published: June 2021

Figs (Ficus carica L.) belong to mulberry tree (Moraceae) which is called fruit of autumn, one of the oldest fruits in the world. Figs have been used as a digestion promoter and a cure for ulcerative inflammation and eruption in Korea. The aim of this study was to develop fermented Fig Vinegar (FV) and to investigate anti‐inflammatory effects of FV. Modification of proteins by long term circulation with glucose leads to the formation of advanced glycation end products (AGEs). Recently, ligation of AGEs play an important role in the development of diabetic complications including atherosclerosis which includes activation, adhesion, and migration of monocytes. FV significantly inhibited MRM‐induced expression of pro‐inflammatory genes including tumor necrosis factor‐α, monocytes chemoattractant protein‐1, interferon‐γ‐inducible protein‐10, and cyclooxygenase‐2 in a dose dependent manner. FV also blocked in iNOS, COX‐2 protein expression level and the attendant PGE2 production. These inhibitory effects may be of benefit for diabetic vascular complications via prevention of oxidative stress in activated human monocytes.
LEPTOMENINGEAL MELANOSIS – A TRUE PEDIATRIC PATHOLOGY/CYTOLOGY CHALLENGE
Authors: Rajaram Veena , Chou Pauline M
Published: June 2021

Leptomeningeal melanosis is a rare entity and often missed on cerebrospinal fluid (CSF) examination. We present two recent cases where the CSF examination was initially reported as negative. A 15year‐old presented with headaches, back pain and an initial diagnosis of an arteriovenous fistula; progressively worsened clinically with concern for increased intracranial pressure. A shunt was placed. The second patient was a 3 year‐old with vomiting and lethargy. Cranial imaging studies showed mildly dilated ventricles with no discrete lesion. CSF analysis in both cases showed blood, hemosiderin‐laden macrophages and rare atypical cells in clusters. These cells were bland with benign appearing nucleus, abundant cytoplasm and cytoplasmic blebs. Rare cells show prominent nucleoli and intranuclear inclusions. They were positive for HMB45 and S‐100. Ultrastructural studies showed melanosomes, consistent with a diagnosis of leptomeningeal melanosis. Review of initial imaging showed diffuse thickening and enhancement of the leptomeninges. In the past 18 years, 7 cases were seen at our institution (5 previously reported in Cancer). In most of these patients the diagnosis was missed at initial CSF evaluation as the neoplastic cells were often overlooked as histiocytes. Therefore, it is important to be aware of this entity as the clinical course is aggressive and uniformly fatal in children.
Activated Notch pathway proteins and FoxG1 are co‐expressed in Medulloblastoma: correlation with phenotype
Authors: Peters L , Mehta V , Chintagumpala M , Dauser R , Adesina A
Published: June 2021

Notch pathway activation has been implicated in the negative regulation of neural differentiation in the developing central nervous sytem. Hes 1 and Hes 5 are downstream effectors of the Notch pathway. FoxG1 forms a transcriptional repressor complex with Hes1 and was recently reported as dysregulated in all histologic types of medulloblastoma. We examined 64 medulloblastomas by IHC for nuclear expression of activated Notch‐1, Hes1, and Hes5. Activated Notch‐1, Hes1, and Hes5 expression were detected in all histologic subtypes. Using Spearman correlation (SC) analysis, there was a positive correlation between the expression of activated Notch1 and Hes1 (SC coeff 0.36, p=0.0035) and Hes 5 (SC coeff 0.433, p<0.001). Expression of FoxG1 and Notch1 also showed a weak, but significant positive correlation (SC coeff 0.273, p=0.028).

Activated Notch1 expression was higher in the anaplastic subtype (ANOVA on Ranks, Dunn's test, H=12.968, p=0.002). Co‐expression of FoxG1 and Notch pathway effectors is consistent with a putative combined role of FoxG1 and activated Notch pathway in the maintenance of an undiferentiated state in medulloblastoma. In view of the reported association of myc amplification with anaplastic progression and the known Notch‐Wnt signaling pathway crosstalk, the significance of our demonstration of a correlation between Notch1 expression and the anaplastic phenotype remains to be explored.
DNA Copy Number Change Analysis for an Anaplastic Ependymoma Case with Recurrence
Authors: Sharma Suash , Mei Yun , Free Alan , Peiper Stephen , Wang Zixuan
Published: June 2021

Anaplastic ependymomas typically occur in the posterior fossa in children. Here we report high resolution genomic copy number studies of an unusual supratentorial anaplastic ependymoma in a 57‐year old man diagnosed in 1998, who developed recurrences, scalp implant in 2006, and ipsilateral neck node metastasis in 2007. High‐resolution comparative genomic hybridization microarray (aCGH) studies using the Affymetrix 250K SNP array was performed on formalin‐fixed, paraffin‐embedded tissues in the first recurrent brain tumor in 2000, the scalp implant, and nodal metastasis, and results compared. Shared changes in all 3 tissues include the loss of chromosome 22q, typically seen in ependymomas; loss of chromosomes 3 and 9; deletions of 11p11.2 and 12q; and amplification of 17q21.31. Brain tumor showed the least overall genomic changes. Loss of 17p13 was seen in brain, but not in implant or metastasis. Shared changes in implant and metastasis include amplification of chromosomes 1q, 5, 7 and deletion of 6p22.3–22.2, 13q12.13 to q34; and homozygous deletion of 9p21.3. This microdeletion spans only ~300 kb and contains the tumor suppressor genes CDKN 2A (P16) and 2B (P15). To conclude, high resolution aCGH effectively detected genomic deletions and amplifications during the evolution of anaplastic ependymoma. These regions include CDKN2A (P16), CDKN2B (P15), and MMP20, which are candidates for aggressive phenotype.
Downregulation of stearoyl‐CoA desaturase in Huntington's disease
Authors: Lingrell Susanne , Lehner Richard , Sipione Simonetta
Published: June 2021

Huntington's disease (HD) is a neurodegenerative disorder characterized by motor, psychiatric and cognitive dysfunction. The disease is caused by the abnormal expansion of a polyglutamine stretch in the N‐terminal region of huntingtin, a ubiquitous protein with unknown function. Mutant huntingtin has been recently shown to interfere with the nuclear localization of the sterol‐regulatory element binding protein (SREBP)‐1, a transcription factor that is key to the expression of enzymes involved in the synthesis of fatty acids. THE OBJECTIVE of this work was to assess whether dysregulation of SREBP‐1 activity affects fatty acid metabolism in HD brains. Using real‐time PCR we have found that expression of stearoyl‐CoA desaturase (SCD), the main mammalian desaturase for the production of monounsaturated fatty acids and a major transcriptional target of SREBP‐1, is down‐regulated in cell and animal models of HD. SCD down‐regulation across HD brain regions mirrors the pattern of neurodegeneration in HD, with the striatum displaying the strongest reduction in SCD expression. Reduced expression of SCD in HD brains is likely to result in an altered ratio between saturated and unsaturated fatty acids, with subsequent perturbations in lipid and membrane composition and altered membrane fluidity, thus representing an important determinant of neural dysfunction in HD. This research is supported by the Huntington Society of Canada and the Canadian Institutes of Health Research.
Mitochondrial immunofluorescence assay as an adjunctive tool in the diagnosis of mitochondrial myopathy
Authors: Rivera‐Zengotita Marie , Mehta Vidya , Craigen Williams , Scaglia Fernando , Bhattacharjee Meenakshi , Wong Lee‐Jun , Adesina Adekunle M.
Published: June 2021

In the modified Walker criteria, the diagnosis of mitochondrial myopathy is supported by decrease in respiratory chain complex (RCC) enzyme activity below the 30th percentile. Due to heteroplasmy, many symptomatic patients with clinical suspicion of mitochondrial disorders do not have biochemical assay levels meeting this criterion, thus requiring sensitive adjunctive assay methods for the detection of reduced RCC protein expression in these patients. We present the results of a mitochondrial immunofluorescence (MIF) assay using frozen muscle tissue for the detection of specific RCC enzyme defects. A total of 31 muscle biopsies from patients with suspected mitochondrial myopathy were analyzed for Complex I, II, III and IV specific proteins by MIF and for enzyme activity by standard biochemical methods. The frequency of abnormal expression of RCC proteins by MIF was Complex I ‐ 66.7%; Complex II ‐ 61.3%; and complex IV ‐ 35.5% compared with 12.9 %, 12.9% and 19.4%, respectively, using the recommended 30th percentile cutoff for reduced enzyme activity. Widespread or regional reduced expression of RCC proteins was seen in many biopsies with biochemical enzyme activities ranging between the 30th and 65th percentiles (Complex I – 54.8%; Complex II – 48.4%; Complex III – 45.2%; Complex IV – 16.1%). We recommend the use of MIF assay as a supplement to current diagnostic methods for the detection of mitochondrial disorders.
Flax seed oil limits bleomycin‐induced pulmonary fibrosis and related right ventricle hypertrophy in rats
Authors: Malberg Ian , Shaffiey Shahab , Herndon Betty , Molteni Agostino , Choi Seojin , Ransom Brian , Baybutt Richard
Published: June 2021

Dietary oils high in α‐linolenic acid have reduced experimental ventricular fibrillation and lowered platelet aggregation, both major causes of cardiac morbidity and mortality. The present trials investigated effects of flax seed oil, high in α‐linolenic acid, on pulmonary hypertension occurring secondarily to lung pathology produced by intratracheal (i.t.) bleomycin in rats. 24 rats received flax seed oil as 0, 5, 10, or 15% of diet for 4 wks, when 12 received bleomycin i.t. and 12 received i.t. saline. At necropsy 2 wks later tissues were fixed for histochemical scoring of pulmonary inflammation and fibrosis. Hearts were dissected, weighed and right ventricle/left ventricle (RV/LV) ratio was determined. Heart and lung scores were compared in bleomycin or saline controls to flax seed oil intake by ANOVA and post‐tests. Septal fibrosis, significantly elevated by bleomycin in the 0% flax seed oil group, was significantly decreased by all doses of the oil, (p<0.001 – 0.05). Right and left ventricle + septum weights were calculated for animals receiving bleomycin compared to the saline controls for each flax seed oil group. The RV/LV bleomycin‐treated groups showed an inverse dose‐response to the percent flax oil in the diet which correlated with the level of fibrosis measured in lungs. These data suggest that moderate levels of dietary flax seed oil limits bleomycin‐induced pulmonary inflammation/fibrosis and that reduction is concurrent with normalization of the RV/LV ratio elevation in the heart.
A role for ERp57 in the regulation of anandamide metabolism
Authors: Yates Marla L. , Barker Eric L.
Published: June 2021

The cannabinoid field is currently an active research area due to a proposed role for endogenous cannabinoids, such as anandamide (AEA), in various physiological processes. AEA is synthesized on‐demand and released into the extracellular space, where it acts as an agonist at both the central and peripheral nervous system cannabinoid receptors CB1 and CB2, respectively. To cease signaling, AEA is transported into cells, where it is metabolized by the enzyme fatty acid amide hydrolase (FAAH1). However, very little is known regarding AEA transport. Our lab has previously identified caveolae‐dependent endocytosis as a mechanism for AEA transport. We recently showed that the protein ERp57 localizes to caveolae during AEA transport. Interestingly, the transcription factor STAT3, which interacts with ERp57 and has been implicated in the regulation of FAAH1 gene expression, localizes to caveolae in its inactive form. We propose that in response to AEA transport, ERp57 and STAT3 work together to up‐regulate FAAH1 gene transcription. Such increases in FAAH1 expression levels have previously been associated with increased AEA transport. We are using RNAi technology to explore the role of ERp57 in STAT3‐regulated FAAH1 transcription. Additionally, we are using various techniques to identify changes in both FAAH1 expression and STAT3 activation in response to AEA uptake. Research funded by NIH Grant R21DA018844.
Cannabinoid1 receptors in the hippocampus mediate the deleterious effects of cannabinoid agonists on spatial memory assessed in the radial arm maze
Authors: Wise Laura Elizabeth , Lichtman Aron H , Thorpe Andrew J
Published: June 2021

The present study tested the hypothesis that the disruption of spatial working memory following cannabinoid (CB) agonist administration is mediated by CB1 receptors in the hippocampus. We first tested whether intrahippocampal administration (IHA) of the CB1 receptor agonist CP 55,940 disrupts memory in the radial arm maze and if this effect is blocked by IHA of the CB1 antagonist rimonabant (RIM). We then assessed whether IHA of RIM blocks the memory disrupting effects of systemically administered (i.p.) CP 55, 940 in the radial arm maze as well as in the tetrad (hypothermia, reduced locomotion, antinociception, and catalepsy). Finally, we assessed whether IHA of RIM blocks the memory disrupting effects of systemically administered (i.p.) THC, the main psychoactive substance in marijuana. It was found that IHA of RIM significantly reduced the number of re‐entry errors in the radial arm maze following IHA or systemic administration of CP 55, 940 but did not the block the effects of CP 55,940 in the tetrad suggesting that IHA of RIM selectively blocks the memory disrupting effects of CP 55, 940. IHA of RIM also reduced errors following systemic THC administration. These findings support the hypothesis that hippocampal CB1 receptors have an important role in mediating the memory impairing effects of cannabinoid agonists. This research was supported by the National Institute on Drug Abuse (DA015683 and T23DA07027).
GABAA receptor alpha6 subunit deficiency prevents development of morphine tolerance
Authors: Meij Johanna Theodora Antoinette , Guo Ning , Kozela Ewa , Behbehani Michael , Kao Winston W.‐Y. , Yu Lei
Published: June 2021

Previously, we compared periaqueductal gray (PAG) samples of morphine‐responsive vs ‐tolerant C57BL/6 mice in a gene array screen, and detected changes in GABAA receptor alpha6 subunit (Gabra6). I.c.v. injection of Gabra6 antisense, but not missense, prevented the increase in ED50 after chronic morphine. By real time RT‐PCR, chronic morphine caused a significant 5‐fold increase in PAG Gabra6 levels in missense‐injected animals, but no change in Gabra6 antisense‐injected mice. We have tested the role of Gabra6 further, in mice with a targeted deletion of the Gabra6 gene (Homanics et al., 1997). 129;B6 Gabra6−/− and their wild‐type littermates received either saline or escalating doses (10–40 mg/kg) of morphine s.c. bid for 3 d. On day 4, mice were tested in a cumulative morphine dose‐tail flick response assay. Chronic morphine induced a rightward shift in the dose‐response curve in WT, but not in Gabra6−/− mice. Thus, PAG Gabra6 expression was increased in morphine‐tolerant mice, whereas Gabra6 antisense blocked both increased expression and development of morphine tolerance. Moreover, Gabra6−/− mice did not develop morphine tolerance. GABAA receptors containing alpha6 have the highest affinity for GABA, are mostly extrasynaptic and may be involved in tonic inhibition. We hypothesize that Gabra6 expression is a compensatory mechanism for altered GABA tone upon chronic morphine treatment.Supported by PHS DA09444 and DA13471 (LY,) and University of Cincinnati Research Council (JTAM)
Effect of Acetyl‐L‐Carnitine on Infarct Size in Multiple Models of Experimental Focal Cerebral Ischemia in Rats
Authors: Jalal Fakhreya Yousuf , Maher Timothy John
Published: June 2021

Acetyl‐L‐carnitine (ALCAR), an acetyl ester of carnitine present in brain, is involved in many aspects of neuronal activity, e.g., energy metabolism, phospholipid synthesis for membrane formation/integrity, and modulation of neurotrophic factors and neurohormones. Aberrant neuronal activity occurs following stroke, and ALCAR has been shown to provide some neuroprotection in a few rat studies. This study determined the neuroprotective effect of ALCAR in multiple rat models of ischemic stroke. Focal cerebral ischemia was induced using distal permanent middle cerebral artery occlusion (MCAO) combined with bilateral carotid artery occlusion (BCCO), proximal permanent MCAO, and both permanent and transient intraluminal suture models. Rats were subjected to distal permanent MCAO combined with BCCO and treated with acute or chronic doses of ALCAR 30 min prior the MCAO. Acute doses of 50, 200, 400 mg/kg I.P. did no affect infarct size, while chronic ALCAR (400mg/kg X 5 days) significantly reduced in infarct size (p< 0.001). Lower doses ALCAR (50 and 200 mg/kg X 5 days) failed to alter infarct size. The effective dose of ALCAR (400 X 5 days) also produced a significant reduction in infarct size in rats subjected to proximal permanent MCAO, permanent, and transient intraluminal suture models. ALCAR might exert its neuroprotection via a host of different mechanistic pathways.
Are adenosine or adrenergic receptors altered in rat brain following chronic aspartame consumption?
Authors: McConnaughey Mona Marie , McConnaughey John
Published: June 2021

The specific aim of this study was to determine if long‐term aspartame administration in rats would cause a change in brain adrenergic or adenosine receptor densities, as a possible biochemical explanation for our previous results showing memory impairment in these animals. Male Sprague Dawley rats (225 g) received either plain tap water or aspartame in the drinking water (250 mg/kg/day) for 4 months. They were then were sacrificed, brains quickly removed and homogenate preparations made and frozen. Receptor numbers were determined using radioligands to label the receptors. Whole brain preparations, from aspartame‐treated rats showed a statistically significant increase in total apparent adenosine A1 receptor numbers when compared to controls (P<0.05). No significant differences were seen between control and treated animals for any of the adrenergic receptors measured. A trend was seen, however, for the alpha1 and alpha 2 receptors to be slightly decreased in the treated animals. More animals and various brain areas need to be studied to further define any differences. Studies such as these are needed to identify possible CNS effects of aspartame and correlate these with observed changes in memory or learning.
Predicting structural requirements of ligands with a multi‐tiered receptor‐based approach to assess estrogen mimicry
Authors: Wittliff James L , Andres Sarah A , Kerr D Alan , Smolenkova Irina A , Bumpus Stefanie B , Le Huy T
Published: June 2021

Diverse chemical features of ligands binding to human estrogen receptor (hERα) illustrate promiscuity of the ligand binding domain (LBD) for interacting with naturally‐occurring and synthetic estrogen mimics. Ligand structural features for LBD recognition and receptor‐induced signal transduction were assessed by competition arrays with Lundon® and GraphPad® software, electrophoretic mobility shift assays with [α32P]Estrogen Response Element sequences and a yeast cell‐based bioassay containing an hERα expression plasmid and a β‐galactosidase reporter plasmid. Activities were modeled with pharmacophoric alignment software GALAHAD™ and DiscoTECH™. Pharmacophores generated were subjected to Comparative Molecular Field and Partial Least Square Analyses to determine quantitative relationships between 3‐D ligand structures and corresponding activities. Using published LBD structures (Leduc et al., PNAS 100:11273‐78, 2003, Ruff et al. unpublished), SurFLEX‐Dock™ software was employed to challenge fitting of 3‐D characteristics of ligands. To improve exposure risk/benefit assessment of candidate molecules, results collectively revealed features of estrogen mimics that promote endocrine disruption. Supported by NIEHS/SBIR #2R44‐ES10076‐02 & Phi Beta Psi Charity Trust. SAA & DAK received IPIBS fellowships and SBB & HTL received Cancer Education Fellowships (NCI 5R25CA44789).
Value of minipig as a recommended second (nonrodent/nonprimate) species for regulatory studies to assess the risk, and relative efficacy of topical and systemic antifungals
Authors: Mainigi Kumar D.
Published: June 2021

Close resemblance of human and minipig skin in hair density, pigmentation, morphology, vasculature, and immunological reactivity makes minipig highly suitable for visual grading of erythema, edema, inflammatory reactions, sensitization, and photo damage. Rabbit is highly sensitive to irritants. Thicker epidermis (50–140μ m) in pig/human absorbs much less than rodents (10–20μ m). Dermal esterases converting prodrug to drug have similar efficiency in pig/human; it is 3 orders of magnitude greater in rat. Similar total hepatic cyt 450 concentration in pig and human is 48 and 93% less than in rat and rabbit, respectively. Properties of pig P450 isozymes are similar to human homologs. Because of longer GI, transit time in pig/man is 4 times greater than dog. Extrapolation of risk from pig to human is probably more realistic. Also similar cardiovascular, pulmonary, urogenital, and GI systems, clinical pathology, moderate growth (2kg/month), more tissue/fluids, and reuse of animals after wash also make minipig desirable for PK/PD, repeat dose, safety pharmacology, and juvenile studies. Hypothetical examples using mini pigs experimentally infected with T. mentagrophytes (dermatophytoses), C. albicans (candidiasis), and A. fumgatus (aspergillosis) to evaluate the safety and efficacy of antifungal combination dermal products shall be discussed. Abstract content are not related to any policy or guideline of FDA
Purification of a Yellow Fluorescent Protein‐Tagged Mu Opioid Receptor and Functional Incorporation into rHDL Particles
Authors: Kuszak Adam John , Whorton Matthew R , Sunahara Roger K
Published: June 2021

Despite extensive characterization of the opioid receptor much is unknown about the biochemical properties of the isolated receptor. Here we present the purification of the mu‐opioid receptor and demonstrate functional reconstitution into phospholipid bilayers. We constructed a human mu‐opioid receptor fused to yellow fluorescent protein at its amino‐terminus (YMOR). Baculovirus‐mediated expression of YMOR in insect cells yields receptors with high affinity [3H]diprenorphine (DPN) binding (Kd=0.5 nM). Dodecylmaltoside‐solubilized (with cholesterol hemisuccinate) YMOR was purified using a series of chromatography steps. Purified YMOR in detergent micelles showed impaired binding to [3H]DPN (Kd=4.14 nM), consistent with the behavior of many GPCRs in detergent. Incorporation of purified YMOR into a phospholipid bilayer in the form of reconstituted high density lipoprotein (rHDL) particles was able to restore high affinity [3H]DPN binding (0.26 nM). YMOR in rHDL bound naltrexone, naloxone and beta‐funaltrexamine with high affinity (Ki = 1.64, 11.9 and 2.8 nM, respectively). DAMGO‐stimulated [35S]GTPgammaS binding to Gi or Go is observed when YMOR is reconstituted into HDL. These data illustrate that the mu‐opioid receptor can be purified to homogeneity and its functional properties restored when incorporated into rHDL particles. This work was supported by NIGMS (GM068603).
Cannabinoid Receptor Interacting Protein (CRIP1a) affects CB1 signaling
Authors: Hardt Tricia Ann , Selley Dana E , Elphick Maurice R. , Lewis Deborah , He Hengjun , Hernandez Jerry
Published: June 2021

Cannabinoid receptor interacting protein 1a (CRIP1a) binds to the last nine amino acids of the cannabinoid (CB1) receptor. Receptor binding studies using human embryonic kidney cells transfected with the CB1 receptor (CB1‐HEK) found that co‐transfection of these cells with CRIP1a (CB1‐HEK‐CRIP1a) did not affect CB1 receptor expression. [35S]GTPγ S binding studies examined the efficacy of various agonists to activate G‐proteins in CB1‐HEK cells with and without CRIP1a transfection. CRIP1a decreased maximal agonist efficacy (Emax) values for the cannabinoids WIN 55,212‐2 (WIN) and CP 55,940 (CP). Emax values for methananamide, levonantradol, and Δ9‐tetrahydrocanabinol (THC) were unaffected. Inhibition of basal CB1 receptor activity by the inverse agonist SR141716A (SR1) was significantly decreased in the presence of CRIP1a. EC50 values of all ligands tested were unaffected. Effects of CRIP1a on CB1 receptor downregulation was examined by incubating CB1‐HEK cells (±CRIP1a co‐transfection) with various cannabinoids and measuring receptor levels via [3H]SR1 saturation binding. CRIP1a inhibited THC‐induced downregulation, but not WIN‐induced downregulation. Downstream signaling studies found no effect of CRIP1a on modulation of adenyly cyclase or MAP kinase activity by maximally‐effective agonist concentrations. A trend toward a decrease in the inverse agonist effects of SR1 was observed. These results suggest that CRIP1a modulates CB1 signaling during acute receptor activation and modulates CB1 receptor downregulation in a ligand‐specific manner. (Funding: NIH, NIDA grant DA‐05274)
Pseudopterosins appear to enhance the wound healing process through an adenosine receptor mediated mechanism
Authors: Moya Claudia E , Jacobs Robert S
Published: June 2021

Pseudopterosins are marine natural products that inhibit phorbol ester (PMA) induced inflammation, inhibit the release of prostaglandins and leukotrienes from macrophages, and also inhibit degranulation of human PMNs. Pseudopterosins exhibit anti‐inflammatory and wound healing properties in several animal models and have shown significant activity in promoting wound healing at donor sites of patients undergoing autologous tissue transplants. At the cellular level, pseudopterosin activity is blocked by pertussis toxin (PTX) pretreatment, indicating that pseudopterosins act upon a G protein or G protein coupled receptor. One of the pseudopterosins, Iso‐pseudopterosin‐E (Iso‐PsE), has recently been shown to bind the adenosine A2A and A3 receptors expressed in HEK‐293 cells with ED50 values of 13 and 6.6 μM, respectively. In these binding experiments the potency of iso‐PsE is low when compared to the specific adenosine A2A agonist (NECA, ED50 = 49 nM) and A3 agonist (IB‐MECA ED 50 = 1.2 nM), but in physiological and in vivo experiments, iso‐PsE is equipotent, and in some cases more potent than NECA and IB‐MECA. In this report we propose the mechanisms by which iso‐PsE binds to these receptors and contributes to the wound healing process. This research is supported by ARMY grant # W81XWH‐06‐1‐0089.
Vascular reactivity to L‐NAME administration in chronic tetraplegia
Authors: La Fountaine Michael , Radulovic Miroslav , Wecht Jill , Cardozo Chrstopher , Spungen Ann , De Meersman Ronald , Bauman William
Published: June 2021

Nitric oxide (NO)‐dependent vascular reactivity, assessed by a change (Δ) in arterial resistance (AR), was evaluated in 5 persons with chronic tetraplegia and 5 age‐matched controls. AR was obtained by venous occlusion plethysmography, and blood pressure was obtained manually. AR was calculated as mean arterial pressure (MAP) divided by the arterial inflow slope. Measures were assessed at baseline (BL) and post intravenous infusion of a NO synthase (NOS) inhibitor, L‐NAME (1 mg/kg), or placebo (normal saline) on separate days. Unpaired t‐tests were used to identify group differences for BL dependent variables (MAP and AR). A one factor ANOVA with a Fisher's post‐hoc analysis was completed on the % Δ [(post‐pre)/pre*100)] of the dependent variables. MAP (74.3 ± 11.3 v. 86.5 ± 8.5, p= 0.01) and AR (19.9 ± 13.6 v. 46.6 ± 30.5, p= 0.02) were lower at BL in the tetraplegia group compared to controls. In the tetraplegia group after L‐NAME: the pressor effect was 58% > placebo (p= 0.001) and 39% > controls (p=0.0008) and AR was 92% > placebo (p=0.004) and 73% > controls (p= 0.018). NOS‐inhibition in the tetraplegia group had an augmented effect to increase MAP, possibly due to the abolished baroreflex capacity. The heightened Δ in AR in those with tetraplegia suggests that enhanced vascular reactivity may be due to a NO‐mediated mechanism and the absence of modulation from interrupted sympathetic innervation. Funds: VA RR&D #B4335V
Olmesartan reduces oxidative stress in the brains of stroke‐prone spontaneously hypertensive rats as measured by an in vivo ESR method
Authors: Araki Shuichiro , Hirooka Yoshitaka , Koga Yasuaki , Ito Koji , Kishi Takuya , Yasukawa Keiji , Utsumi Hideo , Sunagawa Kenji
Published: June 2021

Background : We previously demonstrated that oxidative stress is involved in the neural mechanisms of hypertension. In the present study, we evaluated the effects of anti‐hypertensive treatment with the angiotensin type 1 receptor blocker olmesartan on oxidative stress in the brains of stroke‐prone spontaneously hypertensive rats (SHRSP) using the in vivo electron spin resonance (ESR)/spin probe technique.

Methods and Results : Two groups of 12‐week‐old SHRSP were treated with either olmesartan (10 mg/kg/day) or hydralazine (Hyd, 20 mg/kg/day)/hydrochlorothiazide (HCT, 4.5 mg/kg/day) for 30 days (n=5 for each). Systolic blood pressure decreased after each treatment (151±8 mm Hg [olmesartan] and 156±13 mm Hg [Hyd/HCT], NS). In rats treated with Hyd/HCT, but not those treated with olmesartan, heart rate and urinary norepinephrine excretion increased. The in vivo ESR signal decay rates of the blood‐brain barrier‐permeable spin probe, methoxycarbonyl‐PROXYL, were significantly higher in SHRSP brains compared with age‐matched normotensive Wistar‐Kyoto rat brains (0.121±0.010 vs 0.098±0.011/min, P < 0.01, n=6 for each). Olmesartan attenuated the ESR signal decay rates in SHRSP brains, but Hyd/HCT did not.

Conclusions : These results suggest that olmesartan has an anti‐oxidative effect in SHRSP brains, as measured by an in vivo ESR method, in addition to its blood pressure‐lowering effects.
Probing mineralocorticoid and glucocorticoid receptor physical interaction with specific gene promoters in the mouse heart
Authors: de la Rosa Diego Alvarez , Hernandez‐Diaz Ivan , Giraldez Teresa
Published: June 2021

Expression of mineralocorticoid receptors (MR) in cardiac myocytes is well documented, but it remains unclear whether aldosterone or glucocorticoids are the physiologically relevant ligand in the heart. The use of animal models with inappropriate aldosterone levels for salt status has demonstrated a role for MR in the development of cardiac fibrosis. Similar findings in humans have led to the use of MR antagonists as an important therapeutic measure in the treatment of heart failure. However, very little is known about the molecular mechanisms involved in MR/aldosterone adverse cardiovascular effects or the physiological role that this receptor plays in the heart. MR belongs to the family of steroid nuclear receptors, which function as transcription factors modulating gene expression. Several studies have addressed the effect of MR activation by aldosterone on the transcriptome of mouse heart or cultured cardiac myocytes. However, it is unclear whether aldosterone is the physiological activator of the mouse MR. Therefore, in this study we examined the physical interaction of the MR and the related glucocorticoid receptor (GR) with chromatin in vivo under physiological conditions, regardless of the ligand involved in receptor activation. To that end, we applied the chromatin immunoprecipitation (ChIP) technique to samples obtained from mouse heart. Animals did not receive any treatment and had physiological corticosteroid levels. ChIP products were analyzed for the enrichment of promoter sequences from known corticosteroid target genes. Our data show that both GR and MR are bound to different gene promoters in vivo under physiological conditions. The analysis of ChIP products with qPCR shows preferential binding of GR or MR to specific genes, with significant variations between the different targets tested.
Peripheral Insulin Resistance Accounts for Endothelial Dysfunction in Obesity
Authors: Ali Mohammed Irfan , Marrero Mario B. , Stepp David W.
Published: June 2021

Obesity, peripheral insulin resistance (IR) and endothelial dysfunction (ED) often clinically co‐present, yet mechanistic links remain elusive. Our study tested the hypothesis that peripheral IR is the risk factor underlying ED in obesity. Using cross‐breeding we generated obese, insulin sensitive mice (KdbKPTP) from Leptin receptor mutant (KdbHPTP) mice with IR and mice lacking Protein Tyrosine Phosphatase 1B (PTP1B), a negative modulator of insulin signaling causing IR in muscle and fat (HdbKPTP). Lean mice (HdbHPTP) served as controls. Small mesenteric arteries (SMA, <150 μ m) were analyzed in vitro. Acetylcholine [ACh; +/− L‐NAME, superoxide (SO) dismutase (D)] and Nitroprusside (SNP) reactivity was used to assess endothelium‐dependent/independent dilation. Nitric Oxide (NO) independent dilation was assessed with Papaverine (PV). Maximal dilation to ACh and SNP was less in KdbHPTP than HdbHPTP (50±4, 68±8 vs 70±3, 94±2%, p<0.01) indicating ED and reduced smooth muscle (SM) NO sensitivity in KdbHPTP. SOD improved KdbHPTP dilation to ACh, suggesting a mechanistic role for SO in KdbHPTP ED. HdbKPTP had similar ACh and SNP responses to HdbHPTP. KdbKPTP had less ED versus KdbHPTP (ACh, SNP; 64±4, 94±3 vs 50±4, 68±8%, p<0.01). PV dilation was similar in all mice, ruling out non‐NO dilation as a reason for KdbHPTP ED. We conclude peripheral IR accounts for ED and decreased SM NO sensitivity in obesity.

NIH HL76533
Discovery of Tra2b splicing factor in a novel pathway regulating vascular smooth phenotypic diversity
Authors: Fisher Steven A , Shukla Supriya
Published: June 2021

We are using the regulated splicing of Myosin Phosphatase Targeting subunit 1 (MYPT1) 3′ alternative exon (altex) as a model for the study of vascular smooth muscle (VSM) phenotypic diversity. This exon is skipped in aorta and other large vessels and spliced in the mesenteric resistance artery and portal vein. The splicing modulates in disease models of altered flow and in development. The control mechanisms for the generation of VSM phenotypic diversity are unknown. We identified a consensus binding site for the splicing factor Tra2β (Transformer) 1 in the MYPT1 alt exon. Tra in flies specifies female phenotype by its ability to enhance splicing of a Doublesex altex. The expression of Tra2β correlated precisely with MYPT1 exon inclusion in developmental and disease models, while expressions of other SR family members did not. Tra2β bound to the MYPT1 altexon, and was able to trans‐activate altex splicing from a mini‐gene construct in co‐transfection assays. Mutation or deletion of the Tra2β binding site abrogated these activities. We conclude that Tra2β represents a novel pathway for the activation of the fast (phasic) program of gene expression in the generation of VSM phenotypic diversity. (Support: NIH HL66171 (to S.A.F.)
The effect of β‐agonist on expression and trafficking of β2‐adrenergic receptors with N‐terminal polymorphisms
Authors: Koryakina Yulia A , Jones Stacie M , Kurten Richard C , Cornett Lawrence E
Published: June 2021

The β2‐Adrenergic receptor (β2AR) is an important target for asthma medications. Stimulation of β2AR by agonist inhibits contractile processes and results in bronchodilation. Studies suggest that polymorphisms of the β2AR may act as disease modifiers. We hypothesized that polymorphisms at positions 16 and 27 result in differential expression and trafficking of β2AR following β‐agonist exposure. The functional consequences of these polymorphisms of the β2AR were studied using site‐directed mutagenesis and recombinant expression in HEK293 cells. No differences in receptor density were noted between isoforms after 20 min or 2 h isoproterenol (ISO) treatment in ligand binding assays. However, after 24 h exposure to 1μM ISO, isoforms with Arg16 underwent increased downregulation or reduced receptor density (50.6±2.9% and 70.2±1.7% for R16Q27 and R16E27, respectively) compared to isoforms with Gly16 (27.7±4.2 and 23.2±5.4% for G16Q27 and G16E27, respectively). However, there was no difference between isoforms in co‐localization with Lamp1 (a lysosomal marker) in untreated cells and cells treated with ISO for 24 h. These findings are consistent with an inhibition of β2AR recycling by the Arg16 polymorphism. These results may contribute to understanding the genotype‐specific differences in humans in response to β‐agonist therapy. Support has been provided in part by the Arkansas Biosciences Institute.
Quantitative Measurement of Endothelial Nitric Oxide Synthase in Rat Brain Tissue Homogenates Using an Enzyme‐Linked Immunosorbent Assay Kit
Authors: Czambel R. Kenneth , Kharlamov Alexander , Jones Stephen C.
Published: June 2021

A reliable method of endothelial nitric oxide synthase (eNOS) protein measurement in brain tissue samples would enable the direct study of its regulation and expression. Current eNOS ELISA methods are designed for cell lysates (Aberle et al, Nitric Oxide, 1997, 1: 226). We investigated a commercially available human eNOS ELISA kit (R&D Systems, Minneapolis, MN) for use with rat brain tissue homogenates.

Brain tissue homogenate samples from three normal male Sprague‐Dawley rats were tested for dilutional linearity and spike‐and‐recovery characteristics. Assayed in quadruplicate, a measurable amount of endogenous eNOS was detected in all unadulterated samples (40.0±1.6, 28.2±0.9, and 50.0±1.9 pg/mg wet tissue). All samples were fractionally diluted 9/10, 4/5, 7/10, 3/5, 1/2, and 2/5, with the kit calibrator diluent and displayed good dilutional linearity (linear regression correlation coefficients of 1.0, 0.97, and 0.98). Low spike‐and‐recovery rates (less than 37.5%) were obtained with neat samples; however, prediluting the sample 1/20 before spiking resulted in a 70.3% recovery suggesting eNOS detection is affected by matrix differences between the kit standard and these samples.

With the addition of a correction of recovery, these results suggest the potential usage of this eNOS ELISA kit with rat brain tissue samples.

Support: Pennsylvania Department of Health (Tobacco Settlement Fund)