bluuebunny/crossref_metadata_2025 huggingface dataset:
Proportion of the U.S. Population Consuming a “Low Carbon” Diet
Authors: DOUGLASS JUDITH SPUNGEN , Murphy Mary , Spence Lisa , Schmidt Marlene , DiRienzo Douglas
Published: June 2021

Results of studies on the environmental impacts of food production and transport have suggested that individuals can lower their “carbon footprints” by reducing consumption of animal‐based foods and increasing consumption of plant‐based foods. Specific recommendations for dietary changes to lower environmental impacts have included reduced use of beef, cheese, air‐freighted fish, tropical fruits, and highly processed sweets, snacks and chocolate. These recommendations provide a framework on which to evaluate the impact of adopting a “low carbon diet” on nutrient intakes and measures of diet quality. Using food consumption data reported in NHANES 2003–2006, 2‐day average mean intakes of beef, cheese, tropical fruits (mangos, papayas, bananas, pineapple and starfruit), and the highly processed foods were estimated for 6 age/sex subpopulations of the U.S. population age 9 y and older. Individuals consuming < 75% of the subpopulation mean intakes of beef, < 90% of the mean intakes of cheese and highly processed foods, and < 50% of the mean intakes of tropical fruits were categorized as consuming a diet consistent with “low carbon” goals. Overall, the diets of approximately 11–15% of individuals 9 y and older met the “low carbon” criteria. It will be important to determine the impact of this dietary pattern on nutrient adequacy and overall diet quality. Supported by the National Dairy Council.
Dietitians knowledge of omega‐3 rich foods
Authors: Bangia Deepika , Palmer Debrah M , Adler Audrey , Lewis Nancy M. , Anderson Jennifer , Lohse Barbara
Published: June 2021

Since omega‐3 fatty acids (n‐3s) reduce heart disease risk, and potentially other health problems, e.g., inflammation, mental disorders and poor birth outcomes, it is important for nutrition and health educators to teach the public which foods are rich in n‐3s. To see if dietitians had adequate knowledge of foods rich in n‐3s, surveys were conducted with Registered Dietitians (N=189) in Colorado, Kansas, Nebraska and New Jersey. The majority of the responses were different types of fish and nuts/seeds. Salmon (n=134; 71%), mackerel (n=65; 34%), and tuna (n=62; 33%) were the fish most frequently named, with 97%, 97%, and 95% response certainty respectively. Two other fish were named by more than ten percent of the dietitians, herring (n=23; 12%) (97% response certainty) and sardines (n=25; 13%) (100% response certainty). Walnuts (n=40; 21%) and flaxseeds (n=49; 26%) were also frequently named with high response certainty (98% and 96%). Other foods, e.g., n‐3 rich oils, eggs, wheat germ, and soybeans were also named by some of the dietitians. The survey results show that dietitians had limited knowledge regarding food sources of n‐3s, indicating a need for information resources on this subject for health and nutrition educators. This research was funded by the New Jersey Agricultural Experiment Station and the Rutgers University Food Policy Institute.
Using STELLA to model macronutrient relationships and contributions to weight and body composition change
Authors: Davidson Robert T , Sakita Saori
Published: June 2021

With the increase in obesity rates in the US there is considerable interest in the development of quantitative tools that describe energy balance, weight change and the effects that diet and exercise have on these. We hypothesized that a mathematical description of dietary macronutrient relationships and contributions to macronutrient energy oxidation could be developed that would then calculate changes to body energy stores, body composition and body weight. With the STELLA modeling software and using dose‐response relationships between macronutrient intake and energy oxidation as identified in the published scientific literature, we developed a multi‐compartment, physiologically‐based model which was then validated using different data available from the published scientific literature. This model adequately described the changes in body weight and composition regardless of energy intake level or macronutrient composition. This model may also be used to compare the change in body weight and body composition between different diet regimens that vary in macronutrient composition.
Overexpression of Cyclooxygenase‐2 in the Liver Promotes Hepatocarcinogenesis
Authors: Li Quiying , Han Chang , Xu Lihong , Lim Kyu , Wu Tong
Published: June 2021

This study was designed to investigate the role of hepatocyte COX‐2 in carcinogen‐induced hepatocarcinogenesis. We developed transgenic mice with targeted expression of COX‐2 in the liver by using the albumin promoter‐enhancer driven vector. Male COX‐2 transgenic (Tg) and wild type C57/BL6 mice were subjected to standard diethylnitrosamine (DEN)/phenobarbital tumor induction protocol and monitored for the development of hepatocellular proliferative lesions. The incidence of hepatocellular lesions in COX‐2 Tg mice is significantly higher than in wild type mice (p<0.01). The tumor lesions in COX‐2 Tg mice are significantly larger when compared to wild type littermates at both 16 weeks to 27 weeks. Approximately 40% of COX‐2 Tg mice had multiple macroscopic HCCs 27 weeks after injection of DEN, whereas the wild type counterparts had only microscopic nodules. The COX‐2 Tg mice showed higher nodule multiplicity (1.71±1.27 at 16 weeks, 11.10±6.62 at 27 weeks) than wild type mice (0.25±0.46 at 16 weeks, 1.86±1.75 at 27 weeks) (p<0.01). Western blot analysis of the liver tissues showed higher levels of EGFR, gp130, phosphorylated STAT3, and cyclin D1 in COX‐2 Tg mice than in wild type mice. These results demonstrate that hepatocyte COX‐2 promotes hepatocarcinogenesis and this effect involves activation of EGFR and STAT3 signaling pathways.
Experimental hyperthyroidism treated with acupuncture ‐ metabolomic evaluation of Wistar rats' livers
Authors: Cabral António Jorge Varela , Machado Vanessa Isabel , Nolasco Varão , Tavares Ludgero , Farinha Rodrigo , Carvalho Rui , Cabrita António
Published: June 2021

Hyperthyroidism can be defined as the consequence of production and excessive action of the thyroid hormones. Acupuncture as been proven effective in the treatment of some pathologies, with minimum side effects.

Twenty‐one male Wistar rats were divided into 3 groups: group I – control with no manipulation (Ctrl); group II – received levothyroxine (HT); group III ‐ received levothyroxine and acupuncture (HT+AC). Liver samples were snap frozen in liquid nitrogen and later analyzed by high rotation magic angle NMR spectroscopy (HRMAS) in a in a 14.1 Tesla Varian spectrometer. HRMAS spectral analysis followed by principal component statistical analysis demonstrated that lipids were significantly reduced in the HT group and increased in the HT+AC, the same happening with the aminoacids aspartate and glutamate. Glucose, as well as glycogen were significantly increased in both HT and HT+AC groups.

The results suggest that HT reduces hepatic lipid contents, either by increased mobilization or reduced de novo lipogenesis, and that such reduction is reversed by AC treatment. The increase in glucose content is compatible with either an increase in glycogen breakdown or an increase in gluconeogenesis. Our results show an increase in glycogen content in both HT and HT+AC groups thus supporting the later. Accurate evaluation of lipogenesis and glucose turnover is currently underway using the 2H2O method.
Paracrine induction of fibroblasts by breast cancer cells and their effect on endothelial cell migration
Authors: Rocha Ana Sofia , Pirraco Ana , Soares Raquel
Published: June 2021

The aim of this work was to demonstrate the relevance of the interplay between fibroblasts and breast cancer cells in angiogenesis and metastasis development.

Fibroblasts were treated with conditioned media from the breast cancer cells lines (MCF7 and MDA‐MB‐231). The cultures were evaluated for apoptosis by TUNEL assay, proliferation by BrDU incorporation assay and migration by double‐chamber assay. To examine whether tumor‐induced fibroblasts were able to stimulate endothelium invasion, HUVECs were cultured in a double‐chamber and subjected to conditioned media‐treated fibroblasts.

Apoptosis and proliferation were not affected by conditioned media‐treated fibroblasts as compared to non‐treated cells. However, these cells present higher migration ability. Migration of HUVECs was significantly induced by fibroblasts whenever these were stimulated by breast cancer conditioned media.

Our findings suggest that fibroblast migration is enhanced by breast cancer‐secreted growth factors. In addition, cancer‐induced fibroblasts strongly stimulate endothelial cells migration. These data was corroborated by matrigel plug assay. Taken together, these results reveal the importance of fibroblasts and their primary role in tumor angiogenesis.
Anti‐inflammatory effect of bee venom in amyotrophic lateral sclerosis model
Authors: Yang Eun Jin , Jiang Jing Hua , Lee Sang Min , Lee Myeong Soo , Choi Sun Mi
Published: June 2021

Amyotrophic lateral sclerosis (ALS), paralyzing disorder is characterized by the progressive degeneration and death of motor neurons occurring both as a sporadic and a familial disease. Mutant SOD1(mtSOD1) induces vulnerability of motor neurons through protein misfolding, mitochondrial dysfunction, oxidative damage, cytoskeletal abnormalities and defective axonal transport, excitotoxicity, inadequate growth factor signaling, and neuro inflammation.

Bee venom (BV) known as an Oriental medicine has been reported the evidence in the literature indicated that BV exerts anti‐inflammatory and anti‐nociceptive effects on inflammatory reactions in arthritis and other inflammatory diseases.

The purpose of the present study is to determine whether acupuncture with BV is able to suppress the motor neuron loss and microglial cell action in hG93ASOD1 mouse commonly used model for inherited ALS. BV was treated at the ‘ZuSanLi’ (ST36) acupuncture point in hSODG93A animal model. BV treated animals showed the decrease of micro cell activity and the expression level of phospho‐p38MAPK in spinal cord and brain stem in mutant hSOD1 transgenic mice. Interestingly, BV acupuncture in symptomatic ALS animals improved motor activity and extended survival compared to the control group. Our research suggests a potential functional link between BV and anti‐neuroinflammation in ALS animal model.

the neurotoxicity of mutSOD1 is n

Up to date, no data exist about a link among neuroinflammation, mutSOD1 association in mitochondria and oxidative damage.
Chronic cannabinoid administration lowers viral replication in lymph nodes of SIV infected Rhesus macaques
Authors: Walker Edith M , Amedee Angela , Birke Leslie , Stouwe Curtis Vande , LaCour Nedra , Winsauer Peter , Molina Patricia
Published: June 2021

The primary psychoactive component of marijuana, Δ9‐tetrahydrocannabinol (Δ9‐THC), is used to mitigate AIDS‐associated wasting. Cannabinoid receptors are expressed on cells of the immune system suggesting that chronic Δ9‐THC administration may impact on human immunodeficiency virus progression. Ongoing studies indicate that Δ9‐THC ‐treated, simian immunodeficiency virus (SIV)‐infected rhesus macaques have increased survival and lower plasma viral loads. We hypothesized that chronic Δ9‐THC treatment decreases viral replication by anti‐inflammatory effects at lymphoid tissues. Plasma and lymph nodes obtained at necropsy of SIVmac251‐infected macaques, treated twice daily i.m. with Δ9‐THC (0.32 mg/kg) or vehicle (500μl) were used to quantitate viral load and cytokine levels. Chronic Δ9‐THC treatment resulted in lower plasma viral load (5.28 vs 6.11 log copies of gagRNA/ml plasma), lymph node proviral DNA (1.57 vs 1.99 log copies/10,000 cells) and viral gagRNA (1.14 vs 2.08 log copies/total RNA), irrespective of disease stage. Lymph node content of IL‐1b, IL‐6, IL‐8, and MCP‐1 positively correlated with levels of plasma viral load across all animals (p<0.05). No effect of Δ9‐THC was found on cytokine expression. These results suggest that chronic Δ9‐THC treatment enhances control of viral replication but does not appear to be mediated by decreased inflammation. Supported NIDA‐020419‐01A1.
CORRELATION OF DRβ1*1501, DQβ1*0602 DONOR ALLELIC HAPLOTYPE WITH HUMORAL ANTIBODY EPISODES OF ACUTE RENAL ALLOGRAFT REJECTION
Authors: Mannam Venkat Krishna Reddy , Green Taryn , Cowan Morgan , Goodin Joshua , Lewis Robert , Bigler Steven , Cruse Julius
Published: June 2021

The present investigation was designed to show the effect of molecular HLA class II DR and DQ allelic differences between donor and recipient on humoral antibody rejection identified by C4d peritubular capillary staining. The hypothesis is that expression of the DRβ1*1501, DQβ1*0602 allele in the donor kidney increases the likelihood of humoral antibody rejection. We found that 67 %(n=18) of DR15 and/or DQ6 haplotype donor kidneys induce humoral antibody renal allograft rejection; 35% (n=40) of DR15 and/orDQ6 haplotype donor kidneys failed to induce humoral antibody renal allograft rejection. 41% of C4d+ recipients had donors with DR15; 15% of C4d− recipients had donors with HLA‐DR15. We compared donor haplotype alleles of 4 C4d+ with 6 C4d− recipients by high resolution molecular typing; 3 of 4 C4d+ recipients had a donor with the DRβ1*1501/DQβ1*0602 allele. This allele was absent in all C4d− donors. 35% of C4d+ recipients had 2 DR mismatches when compared to 33% of C4d− recipients. Our results, suggest that the DRβ1*1501, DQβ1*0602 allele in the donor kidney increases the risk of humoral antibody episodes of acute rejection, and signals the need for C4d staining. Future analysis of additional donor and recipient haplotypes will establish whether or not this is a useful predictor of humoral rejection episodes.
Roll of Toll‐like Receptor 9 in Mouse Lung Inflammation in Response to Chicken Barn Air
Authors: Schneberger David , Aulakh Gurpreet , Channabasappa Shankaramurthy , Caldwell Sarah , Singh Baljit
Published: June 2021

Exposure to barn air is an occupational hazard that results in lung dysfunction in barn workers. These respiratory symptoms are typically associated with endotoxin, but within these environments gram negative bacteria may constitute only a small portion of the species and quantity of microorganisms. In contrast, un‐methylated DNA by can be found in all bacteria, most viruses and, and mould. Therefore, we investigated the role of TLR9, which binds to un‐methylated DNA, in barn‐air induced lung inflammation.

First we used immunohistology, immuno‐electron microscopy and in situ hybridization to show expression of TLR9 on bronchial epithelium, alveolar septal cells, and alveolar macrophage of mouse and human lungs. Using a TLR9‐deficient mouse model, these animals were exposed to barn air for 8 hours/day for 1, 5, or 20 days. Examination of bronchiolar lavage and serum against a panel of six common cytokines (IL‐1β, IL‐6, IL‐10, IL‐12, TNF‐α, and IFN‐γ) showed no significant differences after a single day exposure. While TNF‐α (p=0.06) levels in TLR9‐deficient mice were reduced in blood and lavage fluids after 5 and somewhat reduced at 20 days of exposure (p=0.14), IFN‐γ at 5 days(p=0.06) remained reduced after 20 days (p=0.05). Taken together, our data shows expression of TLR9 in mouse and human lungs and that TLR9 may partially contributes to inflammation induced following exposure to chicken barn air.

Grant Funding Source: NSERC Discovery Grant
Seeding of vascular smooth muscle cells confers enhanced pharmacological responses to tissue engineered blood vessels in a large animal model
Authors: Neff Luke P , Tillman Bryan , Yazdani Saami , Machingal Masood Ahammed , Yoo James J , Soker Shay , Bernish Brian , Geary Randolph L , Christ George J
Published: June 2021

These studies evaluated the impact of VSM cell seeding and bioreactor preconditioning on the phenotype and function of tissue engineered blood vessels (TEBV) following 4 months of implantation in vivo. Autologous endothelial progenitor cells (EPC) and vascular smooth muscles cells (VSM) were collected from female sheep and differentiated into mature EC and VSMC in culture. Five TEBV were seeded with both endothelial cells (EC) and VSM, and another 5 grafts seeded with EC only. All grafts were preconditioned to pulsatile flow and physiologic pressure for 2 weeks in a bioreactor. TEBV were implanted in female sheep as an interposition graft of the carotid or femoral artery. The patency rate was 100%, with no aneursymal degeneration or anasomotic stenosis. Vessel wall cells of dual‐seeded grafts had increased immuno‐positive staining to α‐actin (p=0.05) and smooth muscle myosin (p=0.05), as well as increased overall cellularity (p=0.01), when compared to EC only‐seeded grafts. Significantly increased steady‐state maximal contractile responses were observed in the dual‐seeded grafts in response to stimulation with phenylephrine (PE, 10 μM; p=0.01) and 5‐Hydroxytryptamine (5‐HT;10 μM; p=0.01), but not KCl. Maximal contractions to PE and 5‐HT were ≈20% and 15%, respectively, of that observed for the native vessel, respectively. This work represents an important first step toward creation of more native‐like TEBV.
Assay of Heme Oxygenase‐1 through the Direct Measurement of Biliverdin Formation or Iron Release
Authors: Reed James Robert , Backes Wayne L.
Published: June 2021

Heme oxygenase 1 (HO‐1) converts heme to CO, ferrous iron, and biliverdin (BV), receiving electrons used for catalysis from NADPH cytochrome P450 reductase (CPR). Enzymatic studies with the purified, 30 kDa form of HO‐1 routinely use a coupled assay containing biliverdin reductase (BVR) which converts BV to bilirubin (BR). BVR is believed to be required for optimal HO‐1 activity. The goal of this study was to determine if HO‐1 activity could be monitored directly by following BV generation or iron release (using the ferrous iron chelator, ferrozine) in the absence of BVR. Using assays for each of the three end‐products, we found that in the presence of catalase, HO‐1 activity was stimulated and comparable rates were measured in the assays. Absorbance scans revealed characteristic spectra for BR, BV and/or the ferrozene‐iron complex. The optimal conditions were slightly different for the direct and coupled assays. BSA activated the coupled but inhibited the direct assays, and the assays had different pH optima. By measuring the activity of BVR directly using BV as a substrate, these differences were attributed to different enzymatic properties of BVR and HO‐1. Thus, BVR is not needed to measure the activity of HO‐1 when catalase is present. In fact, the factors affecting catalysis by the HO‐1 are better understood using the direct assays because the coupled assay can be influenced by properties of BVR. (Supported by ES004344)
Probing the recognition signal for ubiquitination of suicide‐inactivated neuronal nitric oxide synthase with a C331A mutant of the enzyme
Authors: Clapp Kelly Marie , Peng Hwei‐Ming , Walker Vyvyca J. , Morishima Yoshihiro , Lau Miranda , Osawa Yoichi
Published: June 2021

Nitric oxide synthase (NOS), a cytochrome P450‐like hemoprotein enzyme, catalyzes the synthesis of nitric oxide, a critical signaling molecule in a variety of physiological processes. Our lab has discovered that certain drugs suicide‐inactivate neuronal NOS (nNOS) and lead to the preferential ubiquitination of the inactivated nNOS. We wish to understand how drugs that covalently alter the active site of nNOS labilize nNOS for ubiquitination. A C331A mutant of nNOS was shown to have partially disrupted tetrahydrobiopterin and L‐arginine binding, perhaps similar to a suicide‐inactivated nNOS. We show here that the C331A nNOS was preferentially ubiquitinated by DE52‐retained fraction of reticulocyte lysate, which contains all ubiquitinating enzymes. Moreover, we found that the slowly reversible inhibitor, NG‐nitro‐L‐arginine, but not the inactive D‐isomer, stabilizes and protects both the wild‐type and mutant nNOS from ubiquitination with an IC50 of 0.2 μM and 7.7 μM, respectively. Highly similar results to the in vitro studies were obtained in HEK293T cells after transient transfection of C331A nNOS. The in vitro ubiquitination of nNOS was found to depend on CHIP, an E3 ligase, and hsp70. Thus, it appears that hsp70•CHIP can sense structural changes, most likely small perturbations around the active site, and directs the selective ubiquitination of nNOS. Supported in part by NIH grants GM077430 and DA022354.
Detection of differential splicing of Abcg8 in liver of lactating rats compared to virgin controls using exon arrays
Authors: Athippozhy Antony Thomas , Zhao Tianyong , Coy Donna J , Wooton‐Kee Clavia Ruth , Jungsuwadee Paiboon , Stromberg Arnold J , Vore Mary
Published: June 2021

Lactating dams have an increased demand for cholesterol for secretion into milk, bile acid synthesis and incorporation into cellular membranes. We used Affymetrix Rat Exon 1.0ST® analysis to investigate mRNA expression in liver of lactating (L) vs female control (C) rats. Analysis of splicing variance (ANOSVA) revealed 59 differentially alternatively spliced genes (p<0.001). Abcg5 and Abcg8 function as a heterodimer to export cholesterol into bile, creating a major pathway for cholesterol clearance. Exon array analysis indicated reduced mRNA of Abcg5 and Abcg8 in L vs C, and demonstrated differential alternative splicing of Abcg8 (ANOSVA p=0.0002), but not Abcg5. Screening of calculated expression levels for each exon indicated that exons 6 and 10 were alternatively spliced in L; RT‐PCR confirmed differential splicing. One splice variant detected in L, but not C, showed deletion of part of exons 4 and 9 and all intervening exons by sequencing the RT‐PCR products from the splice variant. Loss of Abcg5/g8 would provide a mechanism for controlling cholesterol loss in bile during lactation. (DK46923)
Isolation of a modulator of the liver specific Organic Anion Transporting Polypeptides (OATPs) 1B1 and 1B3 from Rollinia emarginata Schlecht (Annonaceae)
Authors: Roth Megan , Araya Juan , Timmermann Barbara , Hagenbuch Bruno
Published: June 2021

Organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are liver specific transporters that mediate the uptake of a broad range of drugs into hepatocytes, including statins, antibiotics and many anticancer drugs. Compounds which alter transport by one or both of these OATPs could potentially be used to target drugs to hepatocytes or to improve bioavailability of drugs that are cleared by the liver. In this study, we applied a bioassay guided isolation approach to identify such compounds from the organic extract of Rollinia emarginata Schlecht (Annonaceae). Fractions of the plant extract were screened for effects on OATP1B1‐ and OATP1B3‐mediated transport of 0.1 μM estrone‐3‐sulfate and estradiol‐17β‐glucuronide. A rare compound, quercetin 3‐O‐α‐l‐arabinopyranosyl(1→2) α‐l‐rhamnopyranoside, was isolated that had distinct effects on each OATP. OATP1B1‐mediated transport of both model substrates was strongly inhibited, as was OATP1B3‐mediated transport of estradiol‐17β‐glucuronide. However, OATP1B3‐mediated uptake of estrone‐3‐sulfate was stimulated 4‐ to 5‐fold. Kinetic analysis of this stimulation revealed that the apparent affinity for substrate was increased (Km reduced from 100μM to 10μM), while the Vmax was significantly reduced. These results demonstrate a mechanism through which the hepatic uptake of drug OATP substrates could be stimulated.
Detection of the Free Radicals Produced by β‐amyloid and its Effects on the Acetylcholinesterase Activity
Authors: Rosales‐Hernández Martha Cecilia , Méndez Armando , Zamorano Rafael , Ramírez Daniel
Published: June 2021

One of the most common neurodegenerative diseases, among people older than 65 years, is Alzheimer's disease (AD). The involved neurotransmitter is acetylcholine (ACh), which in turn is hydrolyzed by acetylcholinesterase (AChE). Therefore, evaluate the AChE catalytic activity is very important; which result gives out the alterations in the concentration of the neurotransmitter. The production of the amyloid β‐42 (Aβ‐42) peptide is another process involved in the AD. Aβ form senile plaques which are producers of hydrogen peroxide (H2O2), which reacts with a transition metal (Fe) via Fenton Reaction. That reaction originates some reactive oxygen species (ROS), such as the hydroxyl radical (·OH). Therefore, it is possible that production of ROS modifies the AChE catalytic activity. In this work the catalytic activity of the enzyme was assessed in presence of H2O2 and FeCl2. Later on, H2O2 was replacing by the βA. The presence of the free radical ·OH was determined using electron paramagnetic resonance spectroscopy (EPR) through the presence of spin adduct which is formed by N‐tert‐butyl‐α‐phenylnitrone (PBN) spin‐trap and the ·OH. No modification on AChE catalytic activity were found for high concentrations both H2O2 and de FeCl2. Likewise, the activity of the enzyme was not affected by βA, which could be due to βA did not produce H2O2. This last fact was corroborated by evaluating the catalytic activity of peroxidase and EPR spectroscopy.
Receptor signaling and behavioral properties of EFF0311, a longer‐acting selective full D1 agonist as a potential treatment for Parkinson's disease
Authors: Murthy Vishakantha , Lieu Christopher A , Gowdahalli Krishnegowda , Blake Daniel , Amin Shantu , Subramanian Thyagarajan , Mailman Richard B
Published: June 2021

D1 dopamine full agonists like dihydrexidine (DHX) were previously used to show that D1, but not D2, agonists could match the efficacy of levodopa in the primate MPTP Parkinson's disease (PD) model and in humans with PD. Short duration of action, toxicity, and/or limited bioavailability have prevented a D1 agonist from being approved for clinical use, with only DHX (very short acting) available for limited investigator‐initiated clinical studies. We now report the properties of EFF0311, a DHX analog with greater D1 selectivity and nanomolar affinity for D1 and D5 receptors. EFF0311 had 100% intrinsic activity in stimulating adenylate cyclase via D1 receptors in expressed systems or brain. EFF0311 (relative to quinpirole) was far less potent at D2L‐mediated stimulation of arachidonic acid release than in inhibition of forskolin‐stimulated adenylate cyclase, consistent with functionally selective actions at D2L receptors. In the unilateral 6‐OHDA rat model of PD, EFF0311 effects lasted 3–5 times longer than DHX. These behavioral effects were completely blocked by a D1 antagonist, but not decreased by a D2 antagonist. The properties of EFF0311 (D1 full agonism, longer duration of action, atypical D2 actions) suggest it may be an ideal PD drug candidate.

Grant support: MH082441, MH040537, PA Keystone Innovation Grant (RBM), NS042402 and PA Tobacco settlement fund (TS). RBM has a potential CoI.
The role of D‐serine and other D‐amino acids in a mouse model of amyotrophic lateral sclerosis (ALS)
Authors: Thompson Misty , Reed Ron , Bennett Patrick , Crow John
Published: June 2021

ALS is a fatal disease characterized by selective death of motor neurons (MNs) in the spinal cord. Mutations to Cu, Zn superoxide dismutase (SOD1) have long been known to be a cause of ALS, but the molecular mechanism remains unknown. In the presence of mSOD1, MNs are subjected to increased levels of glutamate excitoxicity via the NMDA receptor, which may be related to increased production of the co‐agonist D‐serine (D‐Ser) from activated glial cells.

As proof of concept, we examined the effects of D‐ser treatment on age of onset, survival, and MN viability in mSOD1 ALS mice. D‐ser treatment resulted in earlier symptom onset by 14.6 ± 0.5 days versus untreated littermates. Paradoxically, D‐ser treatment significantly extended the survival interval (time from onset to sacrifice) by approximately 20 days. The bi‐phasic response to D‐ser may be related to induction of enzymatic D‐ser degradation by D‐amino acid oxidase (DAO). Studies are ongoing to examine spinal cord levels of DAO and serine racemase (produces D‐ser in vivo), both in terms of protein and enzyme activity, levels of D‐amino acids, and overall changes in the proteome (via quantitative 2D‐DIGE proteomics analysis) as a function of disease progression and D‐ser treatment. Our results suggest that the relative concentrations of D‐ser and L‐ser over time may be more important than the absolute concentration of D‐ser alone. Funded by NIH R01 2R01NS040819‐12 (JPC).
L‐pGlu‐(1‐Benzyl)‐L‐His‐L‐ProNH2: a Newer Thyrotropin‐Releasing Hormone Analog with anticonvulsive and neuroprotective properties
Authors: Rajput Satyendra Kumar , Singh Jitendra , Meena Chhuttan , Jain Rahul , Pant Aditya , Sharma Shyam
Published: June 2021

Objective

To study the role of L‐pGlu‐(1‐benzyl)‐L‐His‐L‐ProNH2 (NP‐355) in models of seizures and cellular injury.

Methods

Seizures were induced in mice by pentylenetetrazole (PTZ; 65mg/kg, ip), picrotoxin (PTX; 3.2 mg/kg, sc) and kainic acid (KA; 35mg/kg, ip). Cerebral blood flow (CBF) was measured with laser Doppler in anaesthetized mice. Sodium current was recorded in cultured neonatal rat dorsal root ganglion neurons (DRG) using whole cell patch clamp technique. Glutamate, H2O2 and oxygen glucose deprivation (OGD)‐induced cell injury was studied in PC‐12 cells using MTT assay.

Results

NP‐355 pretreatment showed significant protection in PTZ‐, PTX‐ and KA‐ induced seizures, increased the CBF and its exposure produced depression of sodium current in DRG neurons. In vitro, NP‐355 showed protection from glutamate (15mM) ‐ and H2O2 (0.3mM) ‐ induced cellular injury in a time‐ and concentration‐ dependent manner. The maximal protection was observed after 24 hr of incubation and at 10 μM concentration. The cell viability was increased from 36% to 88% and from 54% to 90% after glutamate and H2O2 treatment in presence of NP‐355. Similarly, a significant difference in cell viability was observed in OGD group with (84%) and without (66 %) treatment. NP‐355 didn't show any abnormal effect on CNS.

Conclusion

NP‐355 is a safe anticonvulsant and neuroprotective agent, also has protective potential against ischemia.
Design of Novel Oxime Reactivators and Direct Characterization of Their Interaction With OP‐ChE Conjugates
Authors: Radic Zoran , Kalisiak Jaroslaw , Cochran Rory , Zhang Limin , Fokin Valery V , Sharpless K Barry , Taylor Palmer
Published: June 2021

Oximes are commonly used nucleophilic reactivators of phosphylated acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Their reactivation rates are several orders of magnitude slower than the rate of ChE inhibition by OPs or rate of acetylcholine hydrolysis, catalyzed by the same enzyme. The rate of oxime reactivation can therefore be substantially enhanced. Using “click chemistry” we prepared more than 800 new oximes and tested them for ability to reactivate phosphylated human ChEs. Interaction constants for three of oximes with native, diethylphosphorylated, VX and cyclosarin phosphonylated and aged human ChEs were determined. Stopped‐flow kinetics of oxime interaction was monitored using quenching of intrinsic tryptophan fluorescence of ChEs as an indicator of oxime binding. We developed mutant AChE resistant to aging and screened the capacity of oximes for reactivation of the mutant. Combination of the mutant enzyme with oxime HI6 proved most efficient for reactivation with rate constant in 10^5 1/(M*min) range being the most efficient ChE based aging resistant catalytic scavenger developed, to date. We also screened our oxime libraries for reactivation of human BuChE and identified candidate oxime reactivators capable of enhancing reactivation rates to an efficient catalytic scavenger of nerve agents.

Supported by U01 NS58046 grant to PT.
NAD+ can induce autophagy of Neuro2a cells and PC12 cells
Authors: Han Jin , Chen Heyu , Wu Zhouyi , Xia Weiliang , Ying Weihai
Published: June 2021

Autophagy is an important form of programmed cell death which could play significant roles in the pathology of multiple diseases. NAD+ is a fundamental molecule in various biological functions. However, it is unknown what is the role of NAD+ in autophagy. The goal of this study is to determine the effects of NAD+ on autophagy using Neuro2a cells and PC12 cells as cell models. We assessed the levels of autophagy by conducting MDC staining assay, real time PCR assay of beclin‐1, and Western blot of LC 3. We found that treatment of 1–10 mM NAD+ produced significant increases in MDC staining and beclin‐1 mRNA levels of Neuro2a cells, as well as significant increases in MDC staining and the ratios of LC 3 II/LC 3 I in PC 12 cells. These studies suggest that NAD+ treatment can induce autophagy of these types of cells. We further found that nicotinamide ‐‐‐ the degradation product of NAD+‐‐‐ could not affect the levels of autophagy, thus arguing against the possibility that NAD+ induces autophagy through generating nicotinamide. In summary, our study indicates that NAD+ can induce autophagy of such cell types as Neuro2a cells and PC12 cells, suggesting a new pathway of inducing autophagy as well as a novel property of NAD+ (Supported by the Key Research Grant of Shanghai Municipal Education Committee, Pujiang Scholar Program Award and a 973 Grant (to W.Y.)).
Anticonvulsant and convulsant effects of cocaine in genetically epilepsy‐prone rats
Authors: Reigel Charles E , Lovering Andrew T
Published: June 2021

Cocaine produces dose‐dependent increases in locomotor activity which can manifest as seizures at high doses. Cocaine is a local anesthetic that also blocks the uptake of norepinephrine (NE), dopamine (DA) and serotonin (5‐HT). Antidepressants that block the uptake of NE and/or 5‐HT produce anticonvulsant effects in genetically epilepsy‐prone rats (GEPRs) at doses below doses producing seizures. The purpose of this study was to determine if cocaine was capable of producing anticonvulsant effects against sound‐induced seizures in the GEPR. Procaine (local anesthetic) imipramine (NE and 5‐HT uptake blockade) and bupropion (DA uptake blockade) effects were also characterized. Anticonvulsant and convulsant dose response curves were determined in moderate seizure (GEPR‐3s) and severe seizure (GEPR‐9s) GEPRs. Effective dose‐50s for anticonvulsant effects and convulsant dose‐50s were determined for all 4 drugs. Cocaine, procaine, imipramine and bupropion all produced anticonvulsant effects in GEPR‐3s and GEPR‐9s at doses lower than doses producing convulsions. These results suggest that cocaine produces anticonvulsant effects at low doses and convulsant effects at high doses through its local anesthetic and NE, DA and 5‐HT uptake blockade mechanisms.
Role of time course and repeated treatment as determinants of morphine effects on intracranial self‐stimulation (ICSS) in rats
Authors: Altarifi Ahmad Adnan , Negus Sidney Stevens
Published: June 2021

Morphine can either increase or decrease rates of intracranial self‐stimulation. The present study evaluated the degree to which these rate‐increasing and rate‐decreasing effects could be dissociated during the time course of acute morphine, during repeated morphine treatment, and during morphine withdrawal. Adult male Sprague‐Dawley rats were fitted with intracranial electrodes targeted at the medial forebrain bundle. Stimulus intensities were individually determined for each rat, and 10 stimulus frequencies (56–158 Hz in 0.05 log increments) were available during each daily session under an FR1 schedule. The primary dependent variable was rate of reinforcement at each frequency. Under control conditions, reinforcement rates increased with frequency. After acute morphine (1–10 mg/kg), rate‐decreasing effects predominated after 10–30 min, and rate‐increasing effects predominated after 100 min. Morphine effects were no longer apparent after 300 min. Repeated morphine (3.2–18 mg/kg × 7 days at each dose) produced tolerance to rate‐decreasing but not rate‐increasing effects. Withdrawal from these doses of repeated morphine did not alter ICSS. These results show that the direction of morphine effects on rates of ICSS is strongly influenced by time course and repeated treatment. Supported by NIH grant R01 NS070715.
Effects of chronic administration of dopamine D2‐like receptor agonists aripiprazole and (−)‐N‐propyl‐norapomorphine on food/cocaine choice in socially housed monkeys
Authors: Czoty Paul W. , Coller Michael , Icenhower Michelle , Nader Michael A.
Published: June 2021

Dopamine D2‐like receptor partial agonists have been suggested as treatments for substance abuse. The goal of this study was to characterize the effects of D2‐like receptor agonists on the relative reinforcing strength of cocaine using a food‐drug choice procedure in socially housed cynomolgus monkeys. In daily self‐administration sessions, monkeys chose between food pellets and cocaine injections (0.003–0.1 mg/kg). The available cocaine dose increased during the session to generate a full dose‐effect curve each day. The high‐efficacy agonist (−)‐N‐propyl‐norapomorphine [(−)‐NPA; 0.001–0.0056 mg/kg] or the lower‐efficacy agonist aripiprazole (ARI; 0.01–0.1 mg/kg) was administered for 5 days; self‐administration was examined on Day 1 and Day 5. At least one ARI dose decreased cocaine choice in most dominant monkeys, but did not affect or increased cocaine choice in subordinates. In contrast, (−)NPA was more effective in decreasing cocaine choice in subordinate monkeys, although increases in cocaine choice were observed in most monkeys. Tolerance was rarely observed across days; in general effects of both drugs were maintained or exacerbated over 5 days. Results suggest that ARI and (−)‐NPA produce qualitatively different effects on the reinforcing strength of cocaine, perhaps due to differing intrinsic efficacies, and that the effects are modulated by social rank. Support: DA12460, DA10584.
The effects of social hierarchy on cocaine reinforcement and brain interactions in male and female monkeys
Authors: Nader Michael A. , Czoty Paul W. , Nader Susan H.
Published: June 2021

Male and female cynomolgus monkeys living in same‐sex social groups form linear hierarchies based on outcomes of fights. Earlier work from our laboratory found that males who became dominant (DOM) had significant increases in dopamine (DA) D2 receptor availability, as measured with PET imaging. There appeared to be behavioral consequences to the increase in D2 signal – DOM monkeys self‐administered (SA) cocaine at lower rates and intakes compared to subordinates (SUB) and the effect of high‐dose cocaine on reinstatement was blunted in DOM compared to SUBs. The present study extended this work to female monkeys. After 3 months of social housing, DOM females also showed a significant (>20%) increase in D2 receptor availability. However, unlike males, DOM females were more sensitive to the reinforcing effects of cocaine, acquiring reinforcement at lower doses than SUBs. In contrast, preliminary data suggest that cocaine pretreatments elicit substantially greater reinstatement in SUB compared to DOM females. There appear to be additional sex differences as a function of social rank related to stress responsiveness and CSF metabolite levels for DA and serotonin. The present findings indicate (1) profound sex differences in the relationship between D2 availability and cocaine reinforcement and (2) a dissociation, in females, between sensitivity to cocaine SA and cocaine‐induced reinstatement. DA17763, DA10584.
Affinity of dopamine receptor antagonists determines the time to maximum effect on cocaine self‐administration
Authors: Fey Brittney K , Tabet Michael R , Norman Mantana K , Tsibulsky Vladimir L , Millard Ronald W , Norman Andrew B
Published: June 2021

The time course of the dopamine receptor antagonist‐induced acceleration of cocaine self‐administration may correspond to the time to achieve maximum fractional occupancy. If so, the time to maximal response (Tmax) will be proportional to antagonist affinity. Male Sprague‐Dawley rats self‐administered cocaine (unit dose of 3 μmol/kg) at regular intervals. The cocaine concentration at the time of each self‐administration (agonist Cmin) was calculated. A series of 9 antagonists spanning a 10,000‐fold range of affinities for D2‐like dopamine receptors was administered i.v. and the effect of each antagonist on the calculated cocaine Cmin was determined.

All of the antagonists accelerated cocaine self‐administration with a concomitant increase in the calculated cocaine Cmin. The antagonist doses that produced approximately equivalent increases in the maximum cocaine Cmin ranged from 0.015 to 4 μmol/kg. The mean Tmax values across this series of antagonists ranged from approximately 5 min to 120 min. Furthermore, the antagonist Tmax values were inversely related to their KD values across this set of antagonists.

These data are consistent with the hypothesis that the agonist Tmax reflects the time course of the increase in antagonist fractional occupancy of D2‐like dopamine receptors. It is concluded that the Tmax is related to antagonist affinity.

Supported by DA018538 and ASPET SURF program.
Serotonin 2C (5‐HT2C) receptor inverse agonists elicit head bob behavior through a dopamine D1 receptor dependent mechanism
Authors: Scarlota Laura , Harvey John A , Aloyo Vincent J
Published: June 2021

Rabbit head bobs and rodent head twitches are behavioral assays of serotonin (5‐HT)2A receptor activation; each behavior can be elicited by DOI (a 5‐HT2A/2C agonist) and attenuated by ketanserin (a 5‐HT2A antagonist). 5‐HT2A receptor antagonists M100907 (M100) and MDL11939 (MDL) also elicit head bobs; however, these effects are distinguishable from DOI‐elicited responses as they are not blocked by ketanserin, suggesting a mechanism other than 5‐HT2A receptor activation. The current study examined the potential role of 5‐HT2C and dopamine (DA) receptors in inducing head bobs. Pre‐administration of the 5‐HT2C antagonist, SB242084 (1umol/kg, sc, 30 min) reduced head bobs elicited by both M100 (2.5umol/kg) and MDL (3umol/kg). Pretreatment of rabbits with the D1 antagonist SCH23390 (0.05umol/kg) also blocked M100 and MDL elicited head bobs. Moreover, administration of a D1 agonist, SKF38393 (9umol/kg) increased head bobs. To further characterize the role of 5‐HT2C receptors, we tested the response to a 5‐HT2C inverse agonist, SB206553. At a dose of 1umol/kg, we found no effect; however, a higher dose (3umol/kg) significantly increased head bobs. Consequently, head bobs may be considered an in vivo measure of 5‐HT2C inverse agonism. Finally, given the role of 5‐HT in DA release, we conclude that SB206553, M100, and MDL all induce the same behavior through 5‐HT2C inverse agonist‐mediated activation of D1 receptors.
Positive GABA modulator‐like effects of 1,1,1‐trichloroethane
Authors: Shelton Keith Larry
Published: June 2021

Despite the magnitude of the problem, our understanding of the neurochemical systems responsible for the abuse‐related behavioral effects of inhalants is lacking. The drug discrimination paradigm in animals has proven to be a valuable tool for understanding the neurotransmitter systems underlying the subjective effects of many drugs and may be a useful procedure for examining inhalants. Sixteen B6SJLF1/J mice were trained to discriminate 10 min of exposure to 12,000 ppm of 1,1,1‐trichloroethane (TCE) vapor from air using a two‐lever, food reinforced, operant procedure. All 16 mice acquired the TCE vapor discrimination. TCE vapor substituted for itself with concentrations of 8000 ppm and greater producing full substitution. Injected doses of the benzodiazepine, midazolam, as well as the barbiturate, pentobarbital, resulted in substitution in most animals. However, the subunit selective benzodiazepine, zaleplon, did not substitute for TCE. The GABA reuptake inhibitor, tiagabine, the GABA transaminase inhibitor, vigabatrin, and the competitive NMDA receptor antagonist, GCS 19755 all failed to produce TCE‐like discriminative stimulus effects. These results are consistent with the hypothesis that the discriminative stimulus of TCE is the result of an indirect, but somewhat selective, positive modulation of GABAA receptor activity. Supported by NIDA grant RO1‐DA020553.
Gambogic acid binds to human CB2 receptors and exhibits potential partial agonist activity
Authors: Rajasekaran Maheswari , Prather Paul L
Published: June 2021

Gambogic acid (GA) induces apoptosis in glioma cells with a capacity to cross the blood brain barrier, thereby representing a potential glioblastoma therapy. Cannabinoid type‐2 receptors (CB2Rs) are upregulated in glioblastomas and their activation also results in impaired tumor growth and cell death. Therefore, it is possible that CB2 receptor activation might contribute to the pro‐apoptotic effects of GA. Employing receptor binding studies in CHO‐hCB2 cells, we report that GA binds to CB2Rs with a Ki of 3.6 ± 0.3 μM. Furthermore, GA dose‐dependently inhibits the activity of adenylyl cyclase (AC) in CHO‐hCB2 cells with an IC50 of 677 ± 274 nM and an Imax of 49 ± 5.3 %. Interestingly, GA also reduces intracellular cAMP levels in wild‐type CHO cells, but with an Imax of only 24 ± 3 %. In CHO‐hCB2 cells treated with pertussis toxin, GA still inhibits AC activity, but with a reduced Imax similar to that observed in CHO‐WT cells. Finally, a concentration (100 nM) of the CB2 antagonist AM630 that is predicted to fully occupy CB2Rs, but does not alter cAMP levels alone, reduces AC inhibition produced by GA to a level observed in CHO‐WT cells. These data suggest that GA produces AC inhibition in CHO‐hCB2 cells via both CB2 and non‐CB2 receptor mediated mechanisms. Therefore, it is concluded that GA binds to CB2Rs with low micromolar affinity and potential partial agonist activity at CB2 receptors might contribute to its anti‐cancer effects.
Lysophosphatidic acid (LPA) receptor subtypes on human gingival and periodontal ligament fibroblasts are regulated by PDGF
Authors: Cerutis D. Roselyn , Headen Karmel V. , Perry Gregory , Parrish Lawrence C. , McVaney Timothy P. , Jordan Curtis S.
Published: June 2021

LPA is a pleiotropic G protein‐coupled lipid mediator involved in many homeostatic and patho‐physiologic aspects of cellular regulation. It binds to LPA1‐6 receptor (LPAR ) subtypes. It is co‐released by activated platelets together with platelet‐derived growth factor (PDGF). We have published that human gingival (GF) and periodontal ligament (PDLF) fibroblasts express LPA1‐3, and that LPA positively regulates their responses to PDGF. In this study, we tested the hypotheses that 1) LPA binds to additional LPAR subtypes on GF and PDLF and 2) PDGF regulates plasma membrane expression of LPARs. Using immunohistochemistry (IHC) with LPAR subtype‐specific antibodies (Abs) we probed for LPA1‐5. Determination of cell‐surface expression of LPARs was done by flow cytometry (FC) with these Abs. Cells were grown in 100‐mm dishes and serum‐starved prior to measuring LPARs by FC. We found that GF and PDLF also express LPA4 and LPA5. PDGF‐BB at10 ng/ml modulated the cell‐surface expression of several LPARs between 2 and 6h post‐treatment. From these results, we conclude that 1) multiple LPAR subtypes modulate the responses of GF and PDLF to LPA, and 2) PDGF‐BB exerts complex regulation of LPARs in these cells. Support: NIH/NIDCR 1 R15 DE016855‐03 (D.R.C).