bluuebunny/crossref_metadata_2025 huggingface dataset:
Tulathromycin promotes phagocytosis and mediates IL‐8, NO, and PGE2 secretion in bovine monocyte‐derived macrophages
Authors: Beatty Jennifer Kathleen , Fischer Carrie D , Morck Douglas W , Lucas Merlyn J , Robb Edward J , Buret Andre G
Published: June 2021

Tulathromycin (TUL) is an antibiotic used in the treatment of bovine respiratory disease, whose efficacy may be due to anti‐inflammatory capabilities. In the lung, macrophage secretion of inflammatory mediators interleukin (IL)‐8, nitric oxide (NO), and prostaglandin E2 (PGE2), as well as phagocytosis apoptotic neutrophils, are imperative mechanisms contributing to the resolution of inflammation. Effects of TUL on modulation of macrophage function have yet to be investigated.

Aim

1) to determine the effects of TUL on neutrophil phagocytosis by bovine monocyte‐derived macrophages (BMDM) and 2) to examine the direct effects of TUL on the expression of cyclo‐oxygenase‐2 (COX‐2) and secreted levels of IL‐8, PGE2, and NO.

Results

Light microscopy suggested that TUL‐induced neutrophil apoptosis is associated with increased neutrophil phagocytosis by BMDM. Western blotting showed TUL increases expression of COX‐2, while ELISA and Greiss reaction showed that TUL decreased secreted levels of IL‐8, and NO and increased levels of PGE2 in lipopolysaccharide (LPS)‐stimulated BMDM.

Conclusion

TUL promotes macrophage phagocytosis of apoptotic neutrophils and modulates COX‐2 protein expression and secreted levels of IL‐8, NO, and PGE2 in LPS‐stimulated BMDM. Together, the findings illustrate novel mechanisms through which an antibiotic may deliver anti‐inflammatory benefits. This work is supported by NSERC.
Erythropoietin improves skeletal muscle microcirculation in mice with sepsis through activation of eNOS
Authors: Kao Raymond , Xenocostas Anargyros , Huang Weixiong , Martin Claudio M. , Rui Tao
Published: June 2021

This study is to explore the potential mechanisms involved in the improvement of microcirculation by the EPO in septic mice.

Methods

Sepsis was induced in mice by intraperitoneal injection of a fecal suspension (12.5 mg/mouse). The septic mice were given rHuEPO (400 U/kg) 18 hrs after sepsis. Capillary perfusion and nicotinamide adenine dinucleotide (NADH) fluorescence were observed using an intravital microscopy. In addition, endothelial cells derived from the skeletal muscle were treated with rHuEPO (5 U/ml) and eNOS activation and activity were assessed.

Results

Septic mice demonstrated microcirculatory dysfunction with a decreased capillary perfusion and an increased tissue NADH fluorescence indicating impaired tissue bioenergetics. In contrast, septic mice treated with rHuEPO resulted in an improvement in the perfused capillary density and decreased muscle NADH fluorescence. However, this beneficial effect of rHuEPO did not occur in septic mice treated with L‐NAME (20 mg/kg) or mice deficient in eNOS. Treatment of endothelial cells with rHuEPO resulted in an increase in eNOS phosphorylation and NO production.

In conclusion

Our results suggest that eNOS plays an important role in mediating the beneficial effect of rHuEPO on microcirculation in this model of sepsis. (Supported by funding from Department of National Defense, Canadian Forces Medical Group).
Cytokine expression in kidney transplant patients with acute allograft rejection
Authors: Warner Roscoe L , Barron Adam , Cibrik Diane M , Johnson Kent J
Published: June 2021

With the development of a 108 protein antibody microarray, we have identified unique expression patterns in the serum of patients with biopsy‐proven acute cellular rejection versus healthy transplant patients. Levels of Gro‐α, IL‐1 RI, IL‐3, IL‐12, IP‐10, Fc‐γ RIIB, MCP‐3 and TGF‐α were significantly increased (p<0.05) in rejecting patients, relative to healthy transplants. Classically in the intense mononuclear inflammatory response of the acute allograft rejection there is an upregulation in the serum of pro‐inflammatory cytokines such as IL‐1, IL‐3, IL‐12, CXC chemokines IP‐10, MCP‐3 and Gro‐α, which are involved in leukocyte migration. Many of these cytokines are present in neutrophils and macrophages and are involved in cell activation and phagocytosis through the activation of NFκB. Conversely, we measured a significant decrease (p<0.05) in IL‐5, MCP‐1, MCP‐ 2, VEGF, MIP‐3αfz PDGF‐AB and TGF‐β2 in rejecting patients relative to the stable transplant patients. Frequently, in acute rejection a shift from TH1 to a TH2 type response is seen in patients with acute rejection as compared to stable transplants, which may explain the subsequent alteration in cytokine levels. These findings, while preliminary may eventually offer biomarkers of acute cellular rejection in patient serum, thus avoiding the need of a kidney biopsy. Work supported in part by NIH RO1 HL0709
Cytokine expression patterns in kidney transplant patients with acute tubular necrosis
Authors: Johnson Kent J , Barron Adam , Cibrik Diane M , Warner Roscoe L
Published: June 2021

Utilizing a custom antibody‐microarray we have identified unique expression patterns in the serum of kidney transplant patients with biopsy‐proven acute tubular necrosis (ATN) versus healthy transplant patients. Levels of Activin A, Fas Ligand, Fcγ RII, IL‐1α, IFN γ, MCP‐1, MCP‐2, MIP‐1α, MIP‐1βfz VEGF, BLC/BCA‐1, GM‐CSF, IP‐10 and IL‐1 RI were significantly increased (p<0.05) in patients with ATN, relative to healthy transplants. A hallmark of ATN is regions of ischemia‐reperfusion (IRI) resulting in localized cellular injury within the tubules. Several current reports demonstrate the production of proinflammatory cytokines (MCP‐1, MCP‐2, MIP‐1α, MIP‐1β) by cells in the renal tubule of patients with ischemic renal failure. In addition, we have seen a significant decrease (p<0.05) in Il‐5, Epo R, MIP‐3α, pro‐Cathepsin S, FGF‐6, TGFβ1, TIMP‐4, VEGF R2 and IL‐18 in ATN patients, relative to the stable transplant patients. These altered cytokine expression in patients with ATN associated renal graft dysfunction may occur at different stages of the disease, necessitating additional patients to confirm our findings. These patterns may eventually provide a biomarker signature of ATN, useful to differentiate patients with renal dysfunction, thus providing a sensitive, non‐invasive method to diagnose kidney injury. Work supported in part by NIH RO1 HL0709.
Mutual Interaction of Prostate Carcinoma And Benign Prostate Hyperplasia Cells With Human Endothelial Cells – PPAR‐gamma Ligands Modulate Parameters of Innate Immunity
Authors: Koenig Wolfgang , Ghebremedhin Beniam , Koenig Brigitte , Koenig Jens‐Ekkehard , Tammer Ina
Published: June 2021

We studied the mutual interaction of human endothelial cells on co‐incubation with prostate carcinoma (PCA, PC3, LNCAP, DU145) and prostate hyperplasia (BPH) cells in the presence of 15d‐PGJ2 and glitazones (troglitazone, ciglitazone). The cells were stimulated with growth factors (VEGF, EGF, HGF, IGF, FGF) at various concentrations (1–100 ng). PPAR‐gamma ligands at various concentrations were added prior to or after co‐incubation with PCA and BPH cells for 24 hours. The release of mediators (IL‐6, IL‐8, PGE2, TNF‐alpha, VEGF) was studied by Elisa. RT‐PCR and Taqman analysis were applied for the analysis of parameters of innate immunity, e. g. p22‐, 47‐, 67‐, gp91‐phox, prostaglandin receptors (EP1‐EP4), toll‐like receptors (TLR2, 4, 9), cox2, retinoid acid receptors [alpha, beta, gamma], IL‐8, CXCR2).

Our results show: 1) Human endothelial cells modulate various parameters of innate immunity on carcinoma as well as BPH cells after co‐incubation. 2) PPAR‐gamma ligands interact differently when given prior or after co‐incubation of tumor cells with human endothelial cells. 3) Differences in pro‐ and antiinflammatory responses depend on the individual cell line as well as the stimulus via differences in cellular signal transduction. The results suggest a potential use of PPAR‐gamma ligands to dampen inflammation and potentially tumor development and tumor spreading.
Depletion of nuclear SOX9 impairs differentiation & facilitates growth of invasive ductal breast carcinoma
Authors: Chakravarty Geetika , Moroz Krystof , Lacey Michelle R , Makridakis Nick M , Agrawal Krishna , Mondal Debasis
Published: June 2021

Invasive breast cancer growth is highly influenced by aberrations in genes controlling normal embryonic development. Recent reports have highlighted the role of Twist, FOXC2, SOX2 and SOX4 in breast cancer (BC) metastasis. But, the role of SOX9, a retinoid inducible gene, in modulating human BC progression remains unknown. SOX9 expression in human BC tissue micro arrays was assessed using immunohistochemistry and cDNA array data from published databases was analyzed for correlation of SOX9 with known poor prognostic parameters of BC. To confirm if deregulation of SOX9 function played a role in BC, genomic analysis of 17q24.3 region was carried out from samples of a large cohort of control and diseased Asian subjects. We found SOX9 expression to be higher in BC cell lines with stem like properties, cells of basal origin, ER negative and high grade human breast tumors. In addition to being associated with poor prognostic indicators of human BC, SOX9 was localized in the cytoplasm of 25–30% human ductal carcinomas and lymph node metastases. Its aberrant cytoplasmic localization or knockdown of its mRNA enhanced the proliferative potential of IDCs. Furthermore, SOX9 locus conferred predisposition to BC amongst Asian women. Our results suggest, SOX9 may functions as a tumor suppressor by promoting differentiation and its deregulation may contribute to the poor clinical outcome associated with invasive breast cancer.
Kallikrein isoforms expressed in skin and GATA3
Authors: Do Son Ngoc , Li LiHua , Komatsu Nahoko , Katsuyama Hironobu , Abe Shuntaro , Higashi Tomomi , Tanii Hideji , Saijoh Kiyofumi
Published: June 2021

Skin is a stiff barrier from the outer environment but, at the same time, desquamation occurs easily in relation not only to constant skin turnover but also to injuries. For desquamation, serine‐proteases/inhibitors balance is important. Among kallikrein families (KLKs), KLK5 and KLK7 are considered to play main role as serine‐proteases. However, when the gene expression level was compared, we found that KLK1 and KLK11 mRNAs were more abundant than KLK5 and KLK7 mRNAs in skin. Therefore, KLK1 promoter was cloned into Luciferase vector to clarify the mechanism of high KLK1 expression. When −957/+36 from the major transcriptional site was transfected, the highest intensity was observed but decreased by −957/−879 depletion. DNA footprinting using nuclear extract from cultured epidermal cell, NHEK, showed the sequence around −924 where GATAs binding site located was protected. RT‐PCR proved that only GATA3 expressed in skin and EMSA using anti‐GATA3 antibody succeeded to supershift the −924 oligonucleotides/nuclear protein complex. Moreover, introduction of GATA3 expression vector to fibroblast succeeded to activate KLK1 promoter and, on the contrary, introduction of GATA3 dominant negative expression vector to NHEK inhibited its activity. Thus, GATA3, which was found to be important for T cell lineage at first, was proved to be one of the major expression regulaters of KLK1 in skin.
Development of novel aryl hydrocarbon receptor antagonists
Authors: Swanson Hollie Isabel , Choi Eun‐Young , Lee Hyosung , Dingle R.W. Cameron , Kim KungBo
Published: June 2021

Increasing evidence indicates that the aryl hydrocarbon receptor (AHR) plays a role in the development and progression of human chronic diseases. Thus, developing appropriate antagonists that target the AHR may not only provide tools to better understand how the AHR signaling pathway modifies human diseases but may ultimately result in novel pharmacological interventions. With this in mind, we have synthesized and characterized a privileged library of putative AHR antagonists that represent derivatives of CH‐223191. Ligand binding analyses have identified at least three derivatives that exhibit equal to or higher ligand binding affinity that the parent compound, CH‐223191. Using reporter assays and mRNA expression analyses, we have found that these CH‐223191 derivatives are not only effective at blocking TCDD‐induced and basal AHR activity, but display minimal agonistic properties. Interestingly, one derivative, AL‐3 appears to synergize with the AHR agonist TCDD with respect to AHR activity. Taken together, these data set the stage for understanding the rules by which optimal AHR antagonists may be developed. This work is supported by ES014849
Regulation of cyp4a31 by AMP‐activated protein kinase and peroxisome proliferator activated receptor alpha
Authors: Bumpus Namandje , Savas Uzen , Johnson Eric F
Published: June 2021

AMP‐activated protein kinase (AMPK) serves as asensor of cellular stress and is an important modulator of lipid and glucose metabolism. The present studies tested the ability of the AMPK activator aminoimidazole‐4‐carboxyamide‐ribonucleoside (AICAR) to regulate murine cyp4 gene expression. Hepatic cyp4 genes were screened for changes in mRNA levels following i.p. injection of eight‐week old C57BL/6 mice with AICAR relative to saline‐treated controls. The largest effect of AICAR 24 hours following injection was on cyp4a31 mRNA expression (3.5‐ and 5.5‐fold increase in males and females, respectively; p<0.01). Chronic injection of males with AICAR resulted in a 2.5‐fold increase of cyp4a31 mRNA (p<0.05) and a 3‐fold increase in lauric acid metabolism (p< 0.01) by liver microsomes prepared from the mice. Since information regarding the regulation of cyp4a31 has yet to be reported we investigated the effect of known regulators of the cyp4a family on the expression of cyp4a31. Cyp4a31 mRNA was markedly increased by treatment of mice with fenofibrate (50‐fold; p<0.01). However, fenofibrate treatment did not increase hepatic cyp4a31 mRNA in PPARα−/− mice. Similarly, preliminary studies demonstrate that cyp4a31 mRNA is not induced by AICAR in PPARα−/− suggesting a role for PPARα in the AICAR‐mediated induction of cyp4a31. (Supported by HD004445 and fellowships from UNCF/Merck and the Fletcher Jones Foundation).
Effect of Cyclosporine A on metabolism and HIF‐1[alpha] induction in APAP toxicity in mice
Authors: Chaudhuri Shubhra , McCullough Sandra S , Hinson Jack A , James Laura P
Published: June 2021

Mitochondrial permeability transition (MPT) is a proposed mechanism for acetaminophen (APAP) toxicity. Cyclosporine A (CYC), an inhibitor of MPT, was previously shown to reduce ATP loss and cytochrome c in APAP toxicity, suggesting that prevention of MPT decreased oxidative stress (J Hep 2005; 42:110–116). In previous work, we found that HIF‐1α was induced in APAP toxicity and hypothesized that HIF‐1α was induced by oxidative stress. Thus, we tested the effect of CYC on HIF‐1α induction in APAP toxicity. CYC of 50 mg/kg (IP) was given after APAP (200 mg/kg IP) and mice were sacrificed at 1, 2 or 4 hr. Mean values for ALT were 2167 IU/L (±379) for APAP/veh mice and 963 IU/L (±317) for APAP/CYC mice at 4 hr (p<0.05). Hepatic glutathione (GSH) depletion was comparable in both groups. No APAP protein adducts were detected at 1 hr and were 40% at 2 and 4 hr in the APAP/CYC group compared to the APAP/veh group (p < 0.05). HIF‐1α expression was reduced in the APAP/CYC mice at 4 hr, compared to the APAP/veh mice. Subsequent studies of HIF‐1α expression were performed using dose ranging of CYC. CYC 20 mg/kg inhibited APAP metabolism. CYC 10 mg/kg was administered to mice after APAP and the mice were sacrificed at 30 or 60 min. ALT, GSH, and APAP protein adduct values were comparable in the two groups of mice at 30 and 60 min. HIF‐1α induction was comparable in the two APAP groups at 30 min. However, HIF‐1α induction was reduced by 40% (p<0.05) in the APAP/CYC mice at 60 min. Thus, in contrast to higher doses, CYC 10 mg/kg did not alter the metabolism of APAP but did reduce HIF‐1α induction. These data support the hypothesis that HIF‐1α induction in the early stages of APAP toxicity is secondary to oxidative stress and the inhibition of MPT. Grant support: DK075936
Induction of intestinal P‐glycoprotein (Pgp) expression and activity by sub‐toxic doses of acetaminophen (AP) in rat intestine and human LS174T cells
Authors: Ghanem Carolina Inés , Arias Agostina , Villanueva Silvina , Novak Analia , Filia María Fernanda , Dellicarpini Griselda , Marín José Juan Garcia , Mottino Aldo Domingo , Rubio Modesto Carlos
Published: June 2021

AP induced liver Pgp expression and activity in rats.

Aim

to study the effect of sub‐toxic doses of AP on intestinal Pgp expression in rats and in a human intestinal cell line.

M&M

Male Wistar rats were injected i.p. for 3 days with 0.2; 0.3; and 0.6 g/kg b.w. of AP (AP group) or vehicle (C group) (n=4). One day later Pgp expression and activity were evaluated by western‐blotting and rhodamine 123 (R123, 15 uM) serosal to mucosal transport in ileum isolated sacs with or without verapamil (V, 100 uM). Intestinal human LS174T cells were exposed to a sub‐toxic dose of AP (5 mM) for 48 hs, then Pgp expression was evaluated by QPCR and R123 efflux was determined by flow citometry with or without V.

Results

Ileum Pgp expression increased in AP vs C (160%, P<0.05). Cumulative R123 secretion was 44% higher in AP group (P<0.05). V inhibited R123 transport in both AP and C. Cells exposed to AP expressed 60% more protein and presented 80% more activity than control cells (P<0.05).

Conclusion

Sub‐toxic doses of AP induced intestinal Pgp expression and activity in rats and in a human intestinal cell line. This suggests potential drug‐drug interactions when AP is co‐administered with other Pgp substrates. Consistently, preliminary studies have shown induction of Pgp in a human intestinal cell line exposed to sub‐toxic concentrations of APAP.
Modulatory effects of lipoic acid and selenium against cadmium‐induced biochemical alteration in testicular steroidogenesis
Authors: Nassar Noha N , EL‐Maraghy Shohda A
Published: June 2021

Exposure to toxic metals, especially Cadmium (Cd), a major environmental pollutant, has become an increasingly recognized source of illness worldwideSince antioxidant therapy provides potential protective role against metal induced testicular damage, hence the current study aimed at investigating the protective effects of lipoic acid and selenium in Cd‐induced testicular damage. Accordingly, male Wistar rats were allocated into four groups (n=8; each). One group received saline and served as control, while the other three groups were given CdCl2 (2mg/kg, i.p. for 28 days) either alone or in combination with (i) lipoic acid (35mg/kg, p.o) or (ii) selenium (as sodium selenite, 0.35mg/kg, p.o) from the first day of Cd administration till the end of the experiment. Serum testosterone, luteinizing hormone and follicular stimulating hormone levels were significantly decreased in the Cd‐exposed rats as well as the activity of testicular key androgenic enzymes such as (Δ53β‐HSD) and 17 β‐HSD as compared the control counterparts. In addition, the activities of testicular marker enzymes were significantly altered in cadmium treated animals. Significant reduction in body weight, testis weight, and testicular antioxidant status was also observed in Cd‐exposed rats. Lipoic acid and selenium significantly restored serum testosterone level and testicular activity of Δ53β‐ HSD, 17 β‐HSD Moreover, we demonstrate alterations in some testicular metal concentrations that were corrected by either selenium or lipoic acid. The biochemical parameters were further confirmed with histopathologial findings. In conclusion, the present study demonstrated the beneficial influences of lipoic acid and selenium in reducing harmful effects of Cd in rats’ testes.
Examination of the Neuroprotective effects of Liposome Encapsulated Curcumin Against Methylmercury induced Neurotoxicity in SH‐SY5Y Cells
Authors: Petroni Daniel H , Mondal Debasis , Tsai Jeffrey , George William J
Published: June 2021

Curcumin has been reported to produce a spectrum of effects, including antioxidant and anti‐inflammatory activity. However, a major limitation to the use of curcumin as a therapeutic agent is its limited absorption and rapid metabolism resulting in low bioavailability. One possible strategy would be to encapsulate curcumin within a liposome, allowing greater penetrance of the drug into the target tissue. This study was conducted to examine the neuroprotective effects of liposomal curcumin (LC) and unencapsulated curcumin (UC) against methylmercury (MeHg), an established neurotoxin. A cell viability tissue culture assay was utilized to examine the neuroprotective effects of both LC and UC. When neuroblastoma (NB) cells were incubated in culture with MeHg (50 nM), cell viability was found to be 61 ± 1.2 % at 48 hours of incubation. The minimal concentration found to produce complete protection against the neurotoxic effects of MeHg for LC and UC was 625nM and 2.5μM, respectively. Blank liposome control did not significantly alter the neurotoxicity of MeHg. These results indicate that curcumin is able to produce a neuroprotective effect against MeHg in NB cells and in the liposomal form, it is significantly more potent than the unencapsulated form. Such a finding supports the hypothesis that LC may have greater penetrance into the cell and thus may be a useful tool to treat methylmercury induced neurological diseases.
Epigenetic Modifications Of Histones Play A Critical Role In Ethanol‐Mediated Enhancement Of FasL Gene Expression And Cell Death In CD4+ T Lymphocytes
Authors: Ghare Smita , Patil Madhuvanti , Joshi‐Barve Swati , McClain Craig , Barve Shirish
Published: June 2021

Alcohol abuse is known to induce immunosuppression involving the depletion of CD4+ T cells; however the mechanisms underlying this alcohol effect are not clearly understood. Recent work from our laboratory showed that ethanol decreases S‐adenosylmethionine (SAM) levels and enhances activation‐induced apoptotic cell death (AICD) in CD4+ T cells. SAM is a critical methyl donor and impacts epigenetic variations involving histone modifications which play a key role in altering chromatin structure and transcriptional activity. Accordingly, the present work was carried out to investigate the immunotoxic effects of ethanol by examining FasL promoter histone modifications in the regulation of FasL gene expression in CD4+ T lymphocytes exposed to alcohol.

Examination of the FasL promoter in CD4+ T cells exposed to ethanol showed increased histone H3 acetylation which is associated with active transcription. In correspondence with increased histone acetylation ethanol was observed to recruit the histone acetyltransferase and transcriptional co‐activator p300. Notably, the histone modifications induced by ethanol enhanced NFAT transcription factor binding and correlated with increased FasL expression. These data strongly support the notion that epigenetic modifications involving histones are critical in ethanol‐induced FasL expression and AICD in CD4+ T cells and ensuing immunosuppression.
Induction of Hypoxia Inducible Factor 1 Alpha (HIF1α) by Caffeic Acid Phenethyl Ester (CAPE) and Caffeic Acid Phenethyl Amide (CAPA) in Mouse Skin Fibroblasts
Authors: Rastogi Ashish , Yang John , Wang Xinyu , Bynum James , Stavchansky Salomon , Bowman Phillip D.
Published: June 2021

Esters and amides of caffeic acid (CA) such as CAPE and CAPA exhibit significant cytoprotection of a variety of cell types from oxidative stress while CA does not. A role for HIF1α in cytoprotection has been described and to better understand the mechanism we investigated skin fibroblasts from the FVB.1 29S6‐Gt(ROSA)26Sortml (HIF1αluc)Kael/J (HIF1αLuc) inbred strain (Jackson Laboratory, Bar Harbor, ME). This mouse (RosaLuc) has been genetically engineered to express luciferase in conjunction with accumulation of HIF1α. RosaLuc mice skin fibroblasts (MSF) were obtained from skin specimens dissociated in 0.1% collagenase for 3 h and cultivated in 10% fetal bovine serum in alpha Minimal Essential Medium. Passages 1–5 were used for these studies and subcultivated into 96‐well multiplates. CAPA was synthesized in the lab and CAPE (Cayman Labs, Ann Arbor, MI) and deferoxamine (DEF), a well known inducer of HIF1α and an iron chelator, were dissolved in DMSO. MSF cells were treated for 6 h with these drugs and assayed for luciferase activity (Promega, Madison, WI). CA was inactive but CAPE and CAPA were significantly better than DEF in producing a luminescent signal from MSF. These results indicate that CAPE and CAPA may directly activate HIF1α by inhibiting prolyl hydroxylase thereby preventing the degradation of nascent HIF1α.
Free radical‐scavenging activity of different solvent extracts from fruit hull of mangosteen
Authors: Ngawhirunpat Tanasait , Opanasopit Praneet , Sitthisombut Chavalit , Kato Atsushi , Adachi Isao
Published: June 2021

Antioxidative and skin protective activities of three extracts, water, methanol, and hexane from the fruit hull of mangosteen and their phenolic constituents such as mangostin, epicatechin, and tannin, were evaluated. The amounts of mangostin, total flavonoid, and total tannin were different among the three extracts, except those of total tannin in methanol and hexane extracts. For the 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) free‐radical scavenging, hydroxyl radical‐scavenging, and inhibition of lipid peroxidation experiment, the GM water extract showed higher activity than the GM methanol extract and GM hexane extract. Mangostin, epicatechin, and tannin also revealed these antioxidant and free radical‐scavenging activities. When added simultaneously with H2O2 (200 micromolar) to keratinocyte cells, the GM water extract (50 mcg/ml), epicatechin (200 micromolar), and tannin (200 micromolar) effectively protected cells from oxidative damage, but the GM methanol extract, GM hexane extract, and mangostin did not. The present study provides the evidence that GM extracts, especially the GM water extract, act as antioxidants and cytoprotective agents against oxidative damage, which is at least partly due to its phenolic compounds in mangosteen.
Beta amyloid 1–42‐induced depressive‐like behavior and decreases in adult neurogenesis are mediated by the phosphodiesderase‐4D (PDE4D) enzyme
Authors: Zhang Han‐Ting , Wu Qiang , Hansen Rolf , Mannan Sunjay , Cheng Yufang , Huang Ying , Hu Wei , Conti Marco , O'Donnell James M
Published: June 2021

Studies have shown that depression may be either a risk factor for Alzheimer's disease (AD) or an early symptom of AD. However, it is not known whether beta amyloid peptide 1–42 (Abeta42), which is mainly responsible for memory loss in AD, plays a role in depression associated with AD. Phosphodiesterase‐4 (PDE4), an enzyme that catalyzes the hydrolysis of cyclic AMP, is involved in the mediation of antidepressant activity and memory. We examined the effects of Abeta42 on behavior in the forced‐swim and tail‐suspension tests in mice. Abeta42 (0.1, 0.4, and 1.6 μg/side), microinfused into bilateral dentate gyri of the mouse hippocampus, increased immobility in a dose‐dependent manner in both tests; it also decreased hippocampal neurogenesis, which is associated with depression. Interestingly, Abeta42 (0.4 μg/side)‐induced depressive‐like effects and decreases in neurogenesis were reversed by deficiency of PDE4D, one of the four PDE4 subtypes. These results suggest that Abeta42 may be an important mediator for depression in AD; disruption of PDE4D appears to be an effective approach to treatment of AD‐associated depression.
Post‐exposure protection against nerve agent‐induced lethality without carbamate pretreatment in guinea pigs
Authors: DeBus Sandra , Spriggs David , Chang Fat‐Chun Tony
Published: June 2021

Managing organophosphorus nerve agent poisoning involves pyridostigmine (PYR) pretreatment followed by post‐exposure oxime/anticholinergic therapy. For military/civilian populations not enrolled in the PYR pretreatment program, however, the post‐exposure therapy offers little, if any, protection. To eliminate the need for pretreatment, we have developed, and efficacy evaluated, a post‐exposure therapy mix consisting of scopolamine (0.5 mg/kg, im), methyl‐atropine (2 mg/kg, im), physostigmine (0.015 mg/kg, im), MMB4 (a bispyridinium oxime; 26.1 mg/kg, im) and phenobarbital (25 mg/kg, ip) against a lethal dose (2×LD50) of either soman (GD), sarin (GB), cyclo‐sarin (GF) or VX in unanesthetized guinea pigs instrumented for concurrent recordings of CNS and cardiorespiratory activities. Results showed that none of the animals tested exhibited seizures, convulsions or signs of anomalous cardiorespiratory activities. Only mild acute cholinergic effects (salivation, dystonia) and phenobarbital's sedative effects were seen during the first 20 min post‐agent. Animals were typically asymptomatic within 20–30 min. All animals survived 24 hrs post nerve agent exposure. Our findings indicate that the therapy mix used in this study was effective not only in antagonizing nerve agent‐induced lethality, but also in protecting the functional integrity of the CNS and cardiorespiratory system.
Alpha‐2A Adrenergic Receptor‐Mediated Antiepileptic and Sedative Effects in Mice
Authors: Azure JoLynn , Poitra Shyleen , Nelson Brian , Goldenstein Brianna , Jurgens Christopher , Weinshenker David , Doze Van
Published: June 2021

Epilepsy, characterized by unprovoked recurrent seizures, is one of the most common neurological disorders in the world. Unfortunately, current treatment options for epilepsy are limited, as many of the antiepileptic drugs have severe side effects such as sedation. Norepinephrine, an endogenous neurotransmitter, has been long known to possess potent antiepileptic properties. The mechanism underlying this particular action is largely unknown. However, recent evidence suggests the antiepileptic effect of norepinephrine is mediated, in part, through alpha‐2 adrenergic receptors (α2ARs). In addition, α2AR agonists such as dexmedetomidine are known to cause sedation. Three subtypes of α2ARs have been cloned: α2A, α2B and α2C. Using transgenic mice and behavioral models for epilepsy (latency to picrotoxin‐induced tonic‐clonic seizures) and sedation (latency to fall off an accelerating rotarod), this study investigated which subtypes of α2ARs are involved in the antiepileptic and sedative effects of the selective α2AR agonist, dexmedetomidine. We found that both of these responses to dexmedetomidine were abolished in α2AAR knockout mice, but not in α2CAR knockout mice. These results may have important implications for the treatment of seizures, as they suggest that only α2AARs are involved in the antiepileptic and sedative effects of norepinephrine. Supported by NSF0851869 and NIH P20RR0167141.
NADPH treatment induces profound necrosis of C6 glioma cells
Authors: Ma Yingxin , Zhao Cuiping , Xia Weiliang , Yan Zhenghui , Sun Lushen , Ying Weihai
Published: June 2021

NADPH is a key factor determining cellular reducing capacity and reductive biosynthesis. However, there has been little study that determines the effects of NADPH on cellular survival and cellular functions. The goal of this current study is to determine the effects of NADPH treatment on survival of cancer cells. Our study using LDH assay showed that treatment of 10 to 1000 micromolar NADPH for 24 hrs or 48 hrs can decrease the survival of C6 glioma cells by 15 to 50% or 5 to 65%, respectively. Trypan blue assays by using a Cell Viability Analyzer have also shown that NADPH treatment can induce significant necrosis of cancer cells. Collectively, our studies have provided first evidence indicating that NADPH can induce necrosis of such cancer cells as C6 glioma cells, suggesting a novel property of NADPH as well as a new means by which cancer cell death can be induced (Supported by the Key Research Grant of Shanghai Municipal Education Committee (to WY), Pujiang Scholar Program Grant (WY), and Shanghai Med‐X Engineering Center Grant of Equipment and Technology of Physical Therapy for Major Diseases (WY)).
Nicotinamide Mononucleotide Can Decrease Rotenone‐Induced Necrosis and Apoptotic Changes of Neuro2a Cells
Authors: Tang Le , Chen Shengdi , Ying Weihai
Published: June 2021

Our laboratory has found that NAD+ can profoundly decrease cell necrosis induced by genotoxic agents, and NAD+ administration can significantly reduce ischemic brain damage. In this study we tested our hypothesis that nicotinamide mononucleotide (NMN)‐‐‐ the precursor of NAD+ ‐‐‐ may also decrease mitochondtial inhibitor‐induced cell death. Our study using LDH assay showed that 0.5 micromolar rotenone induced approximately 40% decreases in the survival of Neuro2a cells, which was significantly attenuated by treatment of 0.1 to 10 mM NMN. Our study applying MTT assay further indicated that 0.1 to 10 mM NMN can attenuate rotenone‐induced decreases in cell viability. We also found that NMN treatment can attenuate the rotenone‐induced nuclear condensation ‐‐‐ a hallmark of apoptosis ‐‐‐ of the cells. Collectively, our study has indicated that NMN can attenue both the rotenine‐induced necrosis and apoptotic changes of Neuro2a cells, suggesting that NMN may be used to decrease mitochondrial inhibition‐induced cell death in neurological diseases (Supported by a Key Research Grant of Shanghai Municipal Scientific Committee 08JC1415400 (WY), Pujiang Scholar Program Grant (WY), and Shanghai Med‐X Engineering Center Grant of Equipment and Technology of Physical Therapy for Major Diseases (WY)).
Eating a high fat chow differentially affects sensitivity of adolescent and adult male rats to cocaine‐induced locomotor activity
Authors: Baladi Michelle G , McGuire Blaine A , France Charles P
Published: June 2021

Dopamine (DA) systems are the target of many drugs of abuse (e.g. cocaine). Many factors, such as age and feeding condition, can alter the behavioral effects of drugs acting on DA systems. For example, adolescent rats are more sensitive than adult rats and food‐restricted rats are more sensitive than free‐feeding rats to some behavioral effects of cocaine; however, much less is known about other dietary manipulations (e.g. high fat diet) on the effects of cocaine. This study examined how access to a high fat chow during adolescence or adulthood impacts sensitivity to the locomotor effects of cocaine. Separate groups of adolescent and adult male Sprague Dawley rats (PND 25 or 85, respectively) had free access to a standard chow (4.4% fat) or to a high fat chow (34.3% fat), or restricted access to a high fat chow (body weight matched to rats eating a standard chow). Regardless of whether adolescent rats had free or restricted access, eating a high fat chow significantly increased sensitivity to cocaine, compared to the effects of cocaine in adolescent rats with free access to a standard chow and compared to adult rats regardless of diet. Thus, adolescence might represent a critical period when feeding conditions influence sensitivity to, and possibly vulnerability to abuse, cocaine or other related drugs.

This work is supported by Senior Scientist Award DA17918.
Wheel running behavior and thermoregulation in mice: effects of MDMA and methamphetamine
Authors: Fantegrossi William E
Published: June 2021

MDMA and methamphetamine (METH) have profound effects on locomotor activity and thermoregulation in rodents. Similarly, spontaneous wheel running behavior increases core temperature in rodents. To study the interaction of these pharmacological and behavioral variables, rediotelemetry probes capable of measuring core temperature were surgically implanted into the peritoneal cavities of male Swiss Wesbter mice. Upon recovery, mice were housed in light‐ and sound‐attenuating chambers containing computer interface equipment for the radiotelemetry probes, a house light (lights off at 13:00 h and on at 01:00 h), a data logger to record ambient temperature, and an exhaust fan. Each mouse had access to ad lib food and water within its home cage, as well as a low profile running wheel, allowing simultaneous collection of wheel running behavior and core temperature across the circadian cycle. Under baseline conditions, ~95% of all wheel running behavior occurred during the subjective dark phase. During wheel running bouts, core temperature increased ~2.5°C, and was tightly correlated with wheel running activity. Injections of MDMA (5 and 10 mg/kg) and METH (1 and 5 mg/kg) were administered at 12:45 h, 15 m prior to the onset of the subjective dark phase and wheel running activity. The effects of these drugs on core temperature and wheel running behavior will be presented. These studies funded, in part, by NIH grant RR020146.
Anti‐conflict effects of triazolam‐pregnanolone combinations in rhesus monkeys
Authors: Fischer Bradford D , Rowlett James K
Published: June 2021

Positive modulators at benzodiazepine (BZ) and neuroactive steroid sites on GABAA receptors interact in an additive or supra‐additive manner depending on the experimental endpoint. The present study examined the combined effects of the BZ triazolam and the neuroactive steroid pregnanolone in a conflict procedure. Rhesus monkeys were trained on a multiple schedule of food presentation in the absence (non‐suppressed responding) and presence of response contingent electric shock (suppressed responding). When administered alone, triazolam and pregnanolone both decreased rates of non‐suppressed responding. Fixed ratio mixtures 1:30, 1:100, and 1:300 triazolam:pregnanolone also decreased rates of non‐suppressed responding, and did so in an additive manner. In contrast, triazolam produced dose‐dependent increases in rates of suppressed responding, whereas pregnanolone failed to produce an effect. Fixed ratio mixtures 1:30, 1:100, and 1:300 triazolam:pregnanolone also produced dose‐dependent increases in rates of suppressed responding. Here, the 1:30 triazolam:pregnanolone mixture resulted in an additive effect; however, the 1:100 and 1:300 mixtures induced significant supra‐additive effects. These results suggest that pregnanolone selectively enhances the anti‐conflict effects of triazolam, which raises the possibility that neuroactive steroids potentiate the anxiolytic effects of BZs.
Individual differences in vulnerability to social stress‐induced cardiac dysfunction in rats
Authors: Wood Susan K , McFadden Kile V , Bhatnagar Seema , Valentino Rita J
Published: June 2021

Social stress precipitates cardiovascular disorders that are likely mediated by exaggerated noradrenergic stimulation. Differing coping strategies result in varying levels of noradrenergic stimulation and may underlie susceptibility to stress‐induced cardiovascular alterations. Repeated social defeat (7 days) using a rat resident‐intruder model resulted in the emergence of two phenotypes. One population (~45%) exhibited a passive response (e.g., freezing) and assumed a defeat posture within a short latency (SL; 197±15s). The other phenotype (~55%) exhibited proactive behaviors (e.g., upright posture) and a longer defeat latency (LL; 438±12s). In vivo telemetry on days 1, 4, and 7 of defeat revealed that LL rats exhibited greater heart rate at rest and during defeat than SL, and resulted in greater cardiac weight. Neuronal activation (cFos) of noradrenergic neurons within the locus coeruleus and dorsal motor vagus was greater in LL rats, while areas mediating the neuroendocrine stress response (paraventricular nucleus, hypothalamus) and freezing behaviors (medial amygdala) were greater in SL rats. SL rats also exhibited more depressive‐like behaviors in the forced swim test. These data suggest that passively coping with social stress renders vulnerability to psychological disorder whereas active coping increases susceptibility to cardiovascular dysfunction. MH 40008, AHA 0825572D, MH06751 to SB
Thrombin mediated PAR1 stimulation results in sustained activation of Rap1 and downstream responses in human 1321N1 astroglioma cells
Authors: Sayyah Jacqueline , Nogal Nekeisha , Dusaban Stephanie , Brown Joan Heller
Published: June 2021

G‐protein coupled receptors that activate RhoA can lead to aberrant cell growth and have been linked to human diseases including cancer. We have shown that in 1321N1 astroglioma cells, stimulation of the PAR1 receptor with thrombin activates RhoA and downstream signals required for mitogenesis. One mediator of thrombin induced mitogenesis is the matricellular protein CCN1 which signals through integrins (Walsh, FASEB J, 2008) and another is phospholipase C epsilon (Citro et al PNAS, 2007). Integrin signaling requires the low molecular weight GTPase Rap1, and PLC epsilon serves as an activator or Rap1. Accordingly we asked whether stimulation of the G12/13 and RhoA coupled PAR1 receptor in 1321N1 cells was coupled to activation of Rap1. Using a pull down assay to assess GTP liganded Rap1, we find that thrombin elicits a robust and sustained increase in Rap1 activation, increasing to 3 fold basal levels and maintained for at least 6 hours. Thrombin stimulation also elicits 4–5 fold increases ERK1/2 and PKD activation which are sustained for at least 6 hours. Our results suggest that thrombin mediated Rap1 activation is associated with sustained ERK1/2 and PKD activation. We are currently exploring the possibility of involvement of RhoA and PLC epsilon in thrombin induced Rap1 activation and the role of Rap1 in sustained ERK and PKD activation induced by thrombin in 1321N1 astroglioma cells.

Supported by NIH grant, GM 36927
Functional selectivity at the serotonin 2A receptor: differential signaling by serotonin and dimethylated tryptamines
Authors: Schmid Cullen L , Bohn Laura M
Published: June 2021

Serotonin and the hallucinogenic metabolites of tryptamines, dimethylated tryptamines, activate the serotonin 2A receptor (5‐HT2AR), which is implicated in numerous mental health disorders. We have previously demonstrated that the 5‐HT2AR is differentially regulated by βarrestin2 (βarr2) depending upon the agonist; here we utilized βarr2 knockout mice to compare signaling profiles in vivo, by assessing the head twitch response (which serves as a behavioral model for selective 5‐HT2AR activation), and in vitro, by assessing kinase activation. We demonstrate that serotonin leads to 5‐HT2AR‐mediated phosphorylation of the serine/threonine kinase AKT in the mouse frontal cortex and in primary neuronal cultures and that the head twitch response occurs in a βarr2 and AKT dependent manner. In contrast, a metabolism‐resistant dimethylated tryptamine, 5‐MeO‐DMT, does not induce phosphorylation of AKT in frontal cortex or in cortical neurons and induces the head twitch response regardless of βarr2 or inhibition of AKT expression. We also show that in the absence of βarr2, the head twitch response is enhanced in response to 5‐MeO‐DMT, suggesting that while βarr2 mediates serotonin signaling at the 5‐HT2AR, it dampens dimethylated tryptamine signaling at this receptor.

Funding: R01 DA025158, NIDA.
Coupling Efficiency of Rhodopsin and Transducin in the Bicelle Mixtures
Authors: Kaya Ali Ilkay , Thaker Tarjani M , Meier Scott M , Preininger Anita M , Iverson Tina M , Hamm Heidi E
Published: June 2021

Although crystal structures of several GPCRs (Rhodopsin, β1‐AR, β2‐AR, α2A‐AR) and G proteins (Gt and Gi) have been determined, the exact molecular mechanism of receptor mediated G protein activation is not fully understood. We are studying the biochemical and structural characteristics of rhodopsin in bicelles (in complex with Gt protein) in order to better understand the mechanism leading to receptor mediated nucleotide release. This study compares the efficiency of various bicelle preparations in stabilizing rhodopsin structure in the absence and presence of G proteins using fluorescence and crystallographic approaches. Our results demonstrate that the extra metarhodopsin II stability increases when soluble rhodopsin is mixed with bicelles. Additionally, the kinetics of extra metarhodopsin II decay of rhodopsin in the bicelle environment is presented, along with receptor mediated G protein activation as measured by GTPγS binding. These data suggest that a membrane‐like structure is required for formation of a stable complex between rhodopsin and G protein, and further, that bicelles provide a native‐like environment for soluble rhodopsin. This may facilitate crystallization trails of the rhodopsin‐G protein complex.
Role of bestrophin‐3 protein in endoplasmic reticulum (ER)‐stress response and its regulation by reactive oxygen species (ROS) and ERK1/2 in kidney proximal tubule cells (PTC).
Authors: Chakraborty Prabir K. , Matchkov Vladimir V. , Thévenod Frank
Published: June 2021

The bestrophin (Best) protein family and its function as chloride channels have not been well characterized. Bestrophin‐3 is suggested to induce Ca2+ activated currents in various tissues. We observed that rat Best‐3 was upregulated by Cd2+ (25μM, 6h) both at the transcriptional and translational levels, in rat kidney PTC. Similar effects were observed with endoplasmic ER‐stress inducing agents, such as thapsigargin (3μM, 6h) and tunicamycin (6μM, 6h) but not with the calcium ionophore A21387 (1μM, 3 and 6h). The upregulation of Best‐3 by Cd2+ was partly dependent on ROS because it could be prevented by the antioxidant α‐tocopherol (100μM). The regulation of Best‐3 expression was also dependent on ERK1/2, which is known to be activated by Cd2+, because the ERK1/2 inhibitor U0126 (10μM) prevented Cd2+‐induced Best‐3 upregulation. Current studies aim to identify the transcription factor/s involved in Best‐3 gene expression. Our findings suggest that Best‐3 plays a role in ER stress response induced by Cd2+and other compounds and is regulated by ROS and ERK1/2 signaling. Funded by DFG TH 345/10‐1.
Structure‐function study of ionotropic glutamate delta‐1 receptor
Authors: Yadav Roopali , Roenfeld Michelle , Scofield Margaret A , Dravid Shashank M
Published: June 2021

Glutamate delta‐1 receptor (delta1) is a member of the ionotropic glutamate receptor family and is expressed in the adult hippocampus and in the inner ear and has been shown to be important in high frequency hearing. Similar to its closest homologue, the delta2 receptor, no agonist induced currents are observed from delta1. The lurcher mutation present in the activation gate in the third transmembrane domain in the SYTANLAAF region in delta2 receptor converts it into a constitutively open cation permeating channel. To understand the structure and function of delta1 receptors we probed the conserved transmembrane domain 3 of the receptor mutating amino‐acids in the activation gate using site‐directed mutagenesis. Employing the two electrode voltage‐clamp technique, four mutants A650C, L652A, A654C and F655A were found to be constitutively open. Leak currents were partially blocked by pentamidine, NASP inhibited 10% of the currents. Studies of the crystal structure of the delta2 receptor agonist binding domain revealed that D‐serine binds to the ABD and inhibited spontaneous currents through delta2Lc. D‐serine inhibited currents through the chimeric delta1‐delta2Lc receptor suggesting residues forming the ABD in delta2 may be conserved in delta1 receptor. Additionally extracellular Ca 2+ may have similar potentiation effect as in Delta2 lurcher. Research support NARSAD.