bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of cholesterol diet and excercise on lipid level, liver function, platelet aggregation and erythrocyte Na+ efflux in rats
Authors: Kang Min Sook , Kim Jung Lye , Kang Young Hee , Kang Jung Sook
Published: June 2021

Present study was conducted to investigate the effects of exercise and cholesterol diet on plasma and liver lipid profile, platelet aggregation, erythrocyte Na efflux and liver index such as GOT and GPT using rats on 0.5% cholesterol diet and/or treadmill for 6 weeks. L.W./B.W. ratio was significantly increased in groups fed cholesterol diet (p<0.01), but exercise tend to decrease this ratio. Plasma total and LDL‐cholesterol was significantly increased and HDL‐cholesterol was decreased in groups with cholesterol diet (p<0.01). Plasma triglyceride was significantly decreased in both groups fed cholesterol diet compared with groups fed normal diet (p<0.01). Exercise decreased plasma triglyceride and showed a significant difference between two groups fed normal diet (p<0.01). Liver total cholesterol and triglyceride was significantly increased in groups fed cholesterol diet (p<0.01), but exercise did not affect on these levels. Intracellular Na and total Na efflux were not different, but Na‐K ATPase tend to increase in groups with exercise. Hematocrit was significantly lower (p<0.05) in group of cholesterol diet without exercise compared with other groups. Platelet aggregation in both the initial slope and the maximum was increased in groups fed cholesterol diet, but not statistically significant. Liver index of both GOT and GPT was significantly increased in groups fed cholesterol diet (p<0.01), and exercise significantly (p<0.01) decreased both GOT and GPT especially in groups fed cholesterol diet when compared with their non exercising partners. This study showed that cholesterol diet increases plasma and liver lipid and GOT and GPT, and exercise improves plasma and liver lipid profile and liver index of GOT and GPT preventing fatty liver.
The role of caveolae‐associated signaling in PCB‐induced tight junction disruption
Authors: Eum Sung Yong , Radakovic Masa , Andras Ibolya Edit , Couraud Pierre‐Olivier , Hennig Bernhard , Toborek Michal
Published: June 2021

Polychlorinated biphenyl (PCB) congeners exhibit a broad range of adverse biological effects including neurotoxicity. The blood‐brain barrier (BBB) is a physical and metabolic barrier separating brain microenvironment from the peripheral circulation and is mainly composed of endothelial cells connected by tight junctions. We previously reported that exposure to highly‐chlorinated PCBs, including 2,2’,4,4’,5,5’‐hexachloro‐biphenyl (PCB153), can decrease expression of tight junction proteins in human brain microvascular endothelial cells (HBMEC) and disrupt the barrier function in an in vitro model of the BBB. However, the mechanisms by which PCBs alter tight junction proteins expression are not completely understood. In the present study, we examined whether caveolae‐associated signaling can contribute to PCB153‐induced dysregulation of tight junction protein expression. Exposure of HBMEC to PCB153 induced phosphorylation of caveolin‐1 which is a major structural and regulatory component of caveolae. In addition, several caveolae‐associated kinases, such as Src family kinase, Ras, and RhoA were activated within 10 min of PCB153 exposure. These data suggest that caveolae and related signaling mechanisms may be involved in PCB‐induced disruption of tight junction proteins and thus contribute to the development of the neurotoxic effects of PCBs in the CNS. Supported by NIH/NIEHS (P42 ES 07380).
PINK1 suppresses 6‐OHDA‐mediated autophagic stress in the neuroblastoma SH‐SY5Y cell line
Authors: Dagda Ruben Karim , Kulich Scott , Chu Charleen
Published: June 2021

PTEN‐induced kinase 1 (PINK1), is a mitochondrially targeted neuroprotective serine/threonine kinase whose mutation has been linked to PARK6 autosomal recessive parkinsonism. We have previously shown that 6‐hydroxydopamine (6‐OHDA) elicits mitochondrial reactive oxygen species (ROS) production which leads to neurodegeneration in vitro (Kulich et al, 2007).

Using several cell survival assays, we show that transient or stable expression of wild‐type PINK1 (PINK1‐WT) in SH‐SY5Y cells partially protects against 6‐OHDA toxicity while transient expression of a kinase‐dead PINK1 mutant (PINK1‐KD) increases 6‐OHDA mediated cell death. Here, we report a set of molecular mechanisms by which PINK1 protects against 6‐OHDA. First, quantification of MitoSOX fluorescence showed that stable expression of PINK1‐WT suppresses 6‐OHDA mediated generation of mitochondrial superoxide. Moreover, stable expression of PINK1‐W.T. increases mitochondrial interconnectivity and blocks 6‐OHDA induced autophagic degradation of mitochondria while transient expression of PINK‐KD had the opposite effect as assessed by confocal microscopy. Thus our data support a model by which PINK1 prevents 6‐OHDA induced autophagic stress by blocking mitochondrial autophagy, increasing mitochondrial interconnectivity and suppressing 6‐OHDA induced production of mitochondrial ROS.
IL‐1β‐induced expression of cytosolic phospholipase A2 in human synovial fibroblast of rheumatoid arthritis
Authors: Chi Pei‐Ling , Yang Chuen‐Mao
Published: June 2021

Arachidonyl‐selective cytosolic phospholipase A2 (cPLA2) is most likely the rate‐liming step in eicosanoid generation. The products of arachidonic acid metabolism are key mediatorsof inflammatory responses in rheumatoid arthritis andyet we do not know the mechanisms of their regulation. We therefore studied the regulation of this pivotal cPLA2 enzyme to generate inflammatory PGE2 in human synovial fibroblasts by pro‐inflammatory cytokines. We demonstrated a dose‐ and time‐dependent expression of cPLA2 protein and mRNA and PGE2 synthesis by IL‐1β which were inhibited by the inhibitors of p38 (SB202190) and JNK (SP600125), respectively. Consistently, IL‐1β‐stimulated phosphorylation of p38 and JNK was attenuated by pretreatment with SB202190 or SP600125, respectively. Transfection with short hairpin RNA (shRNA) of p38 and JNK also abolished IL‐1β‐induced cPLA2 expression, indicating the involvement of p38 and JNK in these responses. Furthermore, IL‐1β‐induced cPLA2 expression and PGE2 synthesis was significantly blocked by a NF‐κB inhibitor Bay11‐7082 and by transfection with dominant negative plasmids of NIK, IKKα or IKKβ. As expected, IL‐1β‐stimulated translocation of NF‐κB into the nucleus was partially blocked by Bay11‐7082, SB202190 or SP600125. Taken together, we conclude that IL‐1β‐induced cPLA2 expression then up‐regulatedinflammatory PGE2 level through p38, JNK, NF‐κB pathways.
New insights into Pseudoxanthoma elasticum pathogenesis by proteome analysis
Authors: Quaglino Daniela , Boraldi Federica , Annovi Giulia , Guerra Deanna , Panico Fulvio , Spaggiari Antonio , De Santis Giorgio , Tiozzo Roberta , Ronchetti Ivonne
Published: June 2021

Pseudoxanthoma elasticum (PXE) is a genetic disorder characterized by progressive calcification of elastic fibers leading to cutaneous, retinal, and cardiovascular lesions. Previous studies on PXE skin biopsies suggested that PXE lesions are characterized by deposition of extracellular matrix constituents and/or fragments that favour mineral precipitates as a result of modified cell behaviour. Very recently, we have demonstrated that PXE fibroblasts suffer from mild chronic oxidative stress due to imbalance between production and degradation of oxidant species. The present study was undertaken with the aim to investigate, by proteome analysis, the protein profile of dermal fibroblasts cultured in vitro from 6 healthy individuals and from 6 PXE patients matched for age and gender. Validation of protein changes was done by FACS and Western Blot. Among the identified proteins that appear to significantly change their expression, the majority of them belong to the endoplasmic reticulum‐related pathways and to the oxidative stress response. Moreover, data indicate that, in PXE, calumenin is significantly over‐expressed, thus contributing to inhibition of gamma‐carboxylase activity and to the reduction of the active form of MGP, a potent inhibitor of soft connective tissue calcification. Work supported by grants from EU (GENESKIN #512117, ELASTAGE #18960) and PXE‐International.
The Role of PKC delta and p190RhoGAP in Actin Filament Stabilization
Authors: Owusu‐Sarfo Akua N , Kwon Eun‐Bee , Settleman Jeffrey , Harrington Elizabeth O
Published: June 2021

Protein kinase C delta (PKCδ) belongs to the PKC family of serine/threonine kinases whose involvement with the actin cytoskeleton and associated protein complexes mediates a myriad of cellular functions, including monolayer permeability. Our published data showed disassembly of focal adhesions (FA), disruption of stress fibers (SF), increased monolayer permeability and decreased RhoA GTPase (RhoA) activity upon inhibition of PKCδ (Harrington E.O. et al, 2005). We also observed binding of PKCδ to p190RhoGAP (p190), a negative regulator of RhoA function. In the current study, we investigated the role of p190 in PKCδ modulation of SF, FA, and monolayer permeability. We found that inhibition of PKCδ in endothelial cells (EC) led to increase in p190 activity. Suppression of p190 in these cells caused a partial loss of SF and reduction in FA, effects which were further accentuated upon inhibition of PKCδ. Interestingly, however, permeability in these cells was not affected by p190 suppression. Examination of p190‐deficient fibroblasts revealed a similar phenotype to that noted in EC treated with PKCδ inhibitors, with a loss of SF and diminished FA formation. Additionally, we observed that RhoA activity is further reduced in p190‐deficient fibroblasts when PKCδ activity is inhibited. Our findings suggest that both p190 and PKCδ may participate in common signaling pathways important in actin filament stabilization.
Characterization of Skeletal Muscle in Osteogenesis Imperfecta Model Mice
Authors: Weber Bettina Anne , Phillips Charlotte L. , Carleton Stephanie M , McCambridge Amanda , Timm David , Ning Jie , Ferreira J Andries , Brown Marybeth
Published: June 2021

Osteogenesis imperfecta (OI), a heritable connective tissue disorder due to mutations in type I procollagen genes, results in bone deformity and fragility. OI model mice (oim) are homozygous for a null mutation in the COL1A2 gene and have significantly reduced bone biomechanical integrity and bone mineral density. Mice heterozygous for the mutation (oim/+) have a phenotype intermediate to that of homozygous (oim/oim) and wild type (+/+) mice. OI patients report reduced muscle capacity and strength, although no systematic evaluation of muscle function and pathology in humans or animal models of OI has been performed. We characterized muscle mass, fiber morphology and contractile generating capacity of oim and oim/+ mice to determine whether muscle weakness is inherent to the disease or due to inactivity. We demonstrated that the contractile generating capacity of hindlimb muscles is not impaired in oim/+ and oim/oim mice relative to +/+ mice; and that muscle cross sectional area and mass/body mass ratios are similar between genotypes, suggesting that oim/+ and oim/oim mice do not have inherent muscle pathology.

NIH/T32 RR0007004‐29, MO Res Brd, Leda J Sears Trust Fdn.
The effect of nicotine on the liver and kidney of prepubertal Sprague Dawley rats
Authors: Al‐Khawaja Khadija M. , Afifi Nahla Maher , Al‐Naemi Hamda
Published: June 2021

The use of tobacco is still rising globally especially between teenagers. Therefore, the effects of early administration of nicotine on the liver and kidney were studied. Forty pre‐pubertal male Sprague Dawley albino rats 4 weeks age weighing 40–65 grams were divided into 4 groups, each of 10 rats. Control groups G1 and G 3 were injected with saline for 1 & 2 weeks respectively, G2 and G4 were injected with nicotine (6.25ng/g) for 1 & 2 weeks respectively. All groups were injected intraperitoneally. Histological studies of the liver showed deleterious effects of nicotine on both liver and kidney in G4. ALT level showed a significant increase (P=0.01) in G4, where a significant increase in AST (P=0.01) was found in both G2 and G4. Urea showed a significant increase (P=0.01) in G4. Lipids however, showed a significant decrease in cholesterol (P=0.01) and HDL‐C (P=0.001) in both G2 and G4, while triglyceride level significantly increased (P<0.05) in G4. X‐ray microanalysis studies showed a significant increase in sodium (P=0.001) in the liver in G4, while magnesium showed a significant decrease (P=0.01) in the kidney in G4. A significant increase in IL‐2 (P<0.05) in both G2 and G4 was detected. However, IL‐6 showed a significant increase (P=0.01) in G4. These results indicate that nicotine exposure increase the risk of damage that occurs in the liver and kidney with increasing the period of exposure to nicotine.
Beta‐Catenin Expression in Pediatric Malignancies
Authors: Cieply Benjamin Walter , Ranganathan Sarangarajan , Monga Satdarshan
Published: June 2021

Wnt/β‐catenin signaling is important in normal liver growth and development where it dictates processes of proliferation, zonation and metabolism. Here we examined pediatric hepatoblastoma (HB), hepatocellular cancer (HCC) and tyrosinemias for expression of β‐catenin, its target Glutamine synthetase (GS) and upstream effector glypican‐3 (GPC3) by immunohistochemistry (IHC). Normal livers displayed membranous β‐catenin, cytoplasmic GS in a single layer of pericentral hepatocytes and no GPC3 expression. 2/9 HCC showed cytoplasmic/nuclear localization, which coincided with increased GS and GPC3. All 10 HB showed strong nuclear and cytoplasmic β‐catenin and increased GPC3, but only 7/10 showed diffuse GS staining. 5/6 livers with tyrosinemia (including 2 with dysplasia and 1 with HCC) showed cytoplasmic β‐catenin, which correlated with diffuse GS and increased GPC3 immunoreactivity. We conclude that significant numbers of HCC, HB and tyrosinemias show aberrant β‐catenin activation, which could be due to increased GPC3, which is known to activate Wnt pathway.
Trace amine‐associated receptor 1 (TAAR1) modulates dopaminergic activity
Authors: Hoener Marius Christian , Lindemann Lothar , Meyer Claas Aiko , Jeanneau Karine , Bradaia Amyaouch , Ozmen Laurence , Bettler Bernhard , Wettstein Joseph G , Borroni Edilio , Moreau Jean‐Luc
Published: June 2021

The recent identification of the trace amine associated receptor 1 (TAAR1) provides an opportunity to dissociate the effects of trace amines on the dopamine transporter from receptor‐mediated effects. In order to separate both effects on a physiological level, a Taar1‐deficient mouse line was generated.

Taar1‐deficient mice displayed increased sensitivity to amphetamine as revealed by enhanced amphetamine‐triggered increases in locomotor activity and augmented striatal release of dopamine as compared to wild‐type animals. Under baseline conditions, locomotion as well as extracellular striatal dopamine levels were similar between Taar1 knock‐out and wild‐type mice. Electrophysiological recordings revealed an elevated spontaneous firing rate of dopaminergic neurons in the ventral tegmental area of Taar1 knock‐out mice. The endogenous TAAR1 agonist p‐tyramine specifically decreased the spike frequency of these neurons in wild‐type but not in Taar1 knock‐out mice, consistent with the prominent expression of Taar1 in the ventral tegmental area.

Taken together, the data reveal TAAR1 as regulator of dopaminergic neurotransmission.
IMPACT OF ADVANCING AGE ON CAFFEINE MEDIATED SENSITIZATION OF CALCIUM RELEASE IN SUPERIOR CERVICAL GANGLION CELLS.
Authors: Behringer Erik J , Vanterpool Conwin K , Pearce William J , Buchholz John N
Published: June 2021

In vascular sympathetic neurons calcium‐induced calcium release (CICR) is mediated by ryanodine receptors (RyR) and contributes to the magnitude of stimulation‐evoked intracellular calcium ([Ca2+]i) transients. Furthermore, this process can be sensitized with RyR agonists such as caffeine. In this study we assessed the contribution of CICR to the magnitude of electric field stimulation (EFS)‐evoked [Ca2+]i transients. We measured EFS‐evoked changes in [Ca2+]i in isolated fura‐2 loaded superior cervical ganglion (SCG) cells, from F‐344 rats aged 6, 12, and 24 months. EFS was delivered over a range of 3 to 24 pulses (3Hz) in the absence and presence of 5mM caffeine. Maximal EFS‐evoked increases in [Ca2+]i significantly increased from 6‐12 months and then declined at 24 months, suggesting that CICR as a component of the EFS‐evoked [Ca2+]i transient declines with age. However, most interestingly, caffeine significantly increased EFS‐evoked [Ca2+]i transients over the entire stimulation range (3–24 pulses) in all age groups. In addition, caffeine decreased the number of pulses to achieve ½ maximal EFS‐evoked [Ca2+]i in all age groups. Taken together, the data suggest that with advancing age although the contribution of CICR to EFS‐evoked [Ca2+]i transients may decline with age, some of this function can be reclaimed in the presence of RyR agonists such as caffeine.

Supported in part by NIH HED P01 #31226
Role for Notch1 signaling in ischemic stroke‐induced endothelial cell dysfunction
Authors: Selvaraj Pradeep Kumar , Mattson Mark P. , Arumugam Thiruma V.
Published: June 2021

Notch signaling plays a major role in cell fate decisions during vascular development in vertebrates. We have recently shown that mice transgenic for antisense Notch and mice treated with gamma‐secretase inhibitors have reduced damage to brain cells and improved functional outcome in a model of focal ischemic stroke. Here we use human microvascular endothelial cells (HMECs), mouse brain endothelial cells (MBECs) and in vivo models of ischemicstroke to elucidate roles for Notch signalingin ischemic stroke‐induced endothelial dysfunction. Preliminary data show that Notch1 and the Notch ligand Jagged expression increase during the first few hours in glucose deprivation (GD) and oxygen and glucose deprivation (OGD) conditions in both HMECs and MBECs. Immunoblot analysis also showed increased expression of Notch intracellular domain (NICD) in response to OGD in MBECs. We also observed increased levels of the Notch signaling‐induced transcription factors (Hes) in response to GD and OGD. Finally, we found that treatment with the gamma‐secretase inhibitor DAPT reduces Notch signaling‐mediated gene expression after OGD condition in MBECs. We are currently studying the role of increased Notch signaling in endothelial dysfunction in a mouse model of ischemic stroke.
Brain‐specific deletion of M3 muscarinic acetylcholine receptor causes dwarfism in mice
Authors: Gautam Dinesh , Jeon Jongrye , Starost Matthew F. , Han Sung‐Jun , Hamdan Fadi , Gavrilova Oksana , Parlow Albert F. , Cui Yinghong , Wess Jurgen
Published: June 2021

The M3 muscarinic acetylcholine (ACh) receptor (M3 mAChR) is widely expressed throughout the brain and in many peripheral organs. Whereas the peripheral M3 mAChRs are known to mediate many of the parasympathetic actions of ACh, the physiological significance of neuronal M3 mAChRs remains unclear. To address this question, we generated mutant mice lacking M3 mAChRs only in neurons and glial cells (‘brain‐M3‐KO mice’). Brain‐M3‐KO mice did not show any significant metabolic alterations, including energy consumption, food intake, and glucose homeostasis. However, adult brain‐M3‐KO mice exhibited a dwarf‐like phenotype (∼10% reduction in body length and ∼20% decrease in body weight, as compared to control mice). Consistent with this phenotype, circulating IGF‐1 and growth hormone (GH) levels were significantly decreased in brain‐M3‐KO mice. Moreover, histological analysis revealed a marked hypoplasia of the anterior pituitary gland of brain‐M3‐KO mice, associated with greatly reduced pituitary GH and prolactin levels. We are currently testing the hypothesis that the lack of M3 mAChRs that normally regulate the activity of hypothalamic GHRH neurons contributes to this phenotype. These studies reveal a critical and unexpected role for neuronal M3 mAChRs in somatic growth and the proliferation of specific anterior pituitary cells.
Molecular and behavioral phenotypes caused by selective disruption of M4 muscarinic acetylcholine receptors in D1 dopamine receptor‐expressing cells
Authors: Jeon Jongrye , Wörtwein Gitta , Fink‐Jensen Anders , Woldbye David P. D. , Schulein Christina , Schütz Günther , Davis Albert A. , Rees Howard D. , Levey Allan I. , Li Cuiling , Deng Chuxia , Cui Yinghong , wess Jürgen
Published: June 2021

The M4 muscarinic acetylcholine (ACh) receptor (M4 mAChR) is enriched in many regions of the forebrain including the striatum. Striatal M4 mAChRs are preferentially found on a subset of striatal projection neurons that express D1 dopamine receptors and give rise to the so‐called direct striato‐nigral pathway. To study the functional roles of these striatal M4 mAChRs, we used Cre/loxP technology to generate mutant mice that lack M4 mAChRs only in D1 receptor‐expressing neurons ('D1‐M4‐KO mice'). Adenylyl cyclase assays with striatal membranes showed that carbachol, a muscarinic agonist, was able to inhibit D1 receptor‐mediated increases in striatal cAMP levels in control mice, but lacked this activity in tissues from D1‐M4‐KO mice. In behavioral studies, D1‐M4‐KO mice showed a significant increase in basal locomotor activity, as compared to control littermates. Furthermore, apomorphine, a non‐selective dopamine receptor agonist, and SKF38393, a D1 receptor selective agonist, induced greater stimulatory locomotor responses in D1‐M4‐KO than in control mice. These studies provide novel insights into the functional roles of striatal M4 mAChRs and their potential involvement in the pathophysiology of various CNS disorders including Parkinson's disease and schizophrenia.
Effect of Lycopus lucidus on high glucose‐induced vascular inflammation in human umbilical vein endothelial cells
Authors: Lee Yun Jung , Kang Dae Gill , Moon Mi Kyoung , Cao Li Hua , Hwang Sun Mi , Kim Jin Sook , Lee Ho Sub
Published: June 2021

High glucose‐induced vascular inflammatory process has been suggested to play a key role in the initiation and progression of atherosclerosis. Lycopus lucidus Turcz. has been used as an Oriental traditional medicine and its crude drug is known to have an anti‐inflammatory effect. Thus we investigated whether the aqueous extract of the leaves of L. lucidus Turcz. (ALT) suppresses vascular inflammatory process induced by high glucose in primary cultured human umbilical vein endothelial cells (HUVEC). Western blot analysis revealed that incubation of HUVEC with high glucose increased cell adhesion molecules (CAMs) expression levels, which were significantly attenuated by pretreatment with ALT in a dose‐dependent manner. The enhanced cell adhesion between monocyte and HUVEC induced by high glucose was also blocked by pretreatment with ALT. High glucose induced hydrogen peroxide production and DCF‐sensitive intracellular reactive oxygen species (ROS) formation were also inhibited by pretreatment with ALT. In addition, ALT suppressed the translocation and promoter transcriptional activity of NF‐κB increased in high glucose condition. Taken together, the present data suggested that ALT could suppress high glucose‐induced vascular inflammatory process, which may be closely related with the inhibition of ROS and NF‐κB activation in HUVEC.
Active metabolite of vitamin D acutely reduces endothelium‐dependent contractions in the isolated SHR aorta
Authors: Wong Michael SK , Man Ricky YK , Vanhoutte Paul M
Published: June 2021

Recent studies proved that vitamin D has cardiovascular effects besides regulating calcium homeostasis.

Objectives:

To study the effect of calcitriol (1,25‐dihydroxyvitamin D3) on endothelium‐dependent responses of the aorta of spontaneously hypertensive rats (SHR).

Methods:

Rings of aortae were suspended in organ chambers for isometric force measurement. Agonist‐induced endothelium‐dependent and independent responses as well as prostacyclin release were measured in the absence or presence of calcitriol. Cytosolic calcium concentration was monitored by confocal microscopy after incubation with fluorescent dyes, Fluo‐4 and Fura‐red.

Results:

Acetylcholine‐induced relaxation was not affected by administration of calcitriol while the acetylcholine‐ and ATP‐induced endothelium‐dependent contractions were reduced compared to those obtained in the absence of the drug. This effect was not present if the calcium ionophore A23187 was used as agonist. Acetylcholine‐ but not A23187‐induced release of prostacylin was reduced by acute administration of calcitriol. Exposure to calcitriol reduced the increase in cytosolic free calcium concentration caused by acetylcholine in the endothelial cells.

Conclusions:

This study shows that calcitriol can modulate vascular tone by reducing the release of endothelium‐derived contracting factor(s). (Partly supported by the CeDAR Foundation)
Inhibition of sphingosine kinase induces vasoconstriction in isolated carotid arteries obtained from spontaneous hypertensive but not normotensive rats
Authors: Spijkers Leon , Mathy Marie‐Jeanne , Michel Martin , Alewijnse Astrid , Peters Stephan
Published: June 2021

Ceramide, sphingosine and sphingosine‐1‐phosphate (S1P) are signaling molecules generated from membrane phospholipids by a concerted action of several enzymes. These sphingomyelin metabolites (SM) have growth regulating properties; while S1P is implicated in growth stimulation, ceramide and sphingosine (the precursors of S1P) induce apoptosis. In addition, SM have vasoactive properties. For instance S1P, synthesized by sphingosine kinase, can increase NO production in endothelial cells and thereby induce vasodilation. Hypertension is a disease state characterized by endothelial dysfunction (decreased NO bioavailability) and vascular growth (remodeling). Therefore we investigated whether hypertension alters the vasoactive properties of locally produced SM. Inhibition of sphingosine kinase by means of dimethylsphingosine (DMS, 10 μM) induced a biphasic and transient contraction in isolated rat carotid arteries from spontaneous hypertensive but not normotensive animals. This contraction was endothelium and eicosanoid‐dependent since removal of the endothelium and inhibition of cyclooxygenase completely prevented DMS‐induced constriction. Alterations in sphingolipid metabolism may thus contribute to a disturbed regulation of vascular tone during hypertension.

This research was performed within the framework of project T2‐108 of the Dutch Top Institute Pharma.
HIV protease inhibitor‐induced inflammatory response is coupled to ER Stress and MAPKs signaling pathways in macrophages
Authors: Chen Li , Jarujaron Sirikalaya , Wu Xudong , Gurley Emily , Studer Elaine , Hylemon Phillip B , Pandak William M , Cao Risheng , Zhang Luyong , Zhou Huiping
Published: June 2021

Macrophages are the key players in the inflammatory response. Our previous studies have shown that HIV protease inhibitors (PIs) induce ER stress and increase TNF‐¦Á and IL‐6 expression in macrophages. However, the underlying signaling mechanisms remain unclear. The aim of present study was to determine if MAPKs are involved in HIV‐PI‐induced inflammatory response in macrophages. J774A.1 cells and primary mouse peritoneal macrophages isolated from wild type and CHOP knock out mice were used in this study. Cells were treated with individual HIV PI. The mRNA and protein levels of TNF‐¦Á and IL‐6 were measured by real‐time RT‐PCR and ELISA, respectively. Activation of MAPKs was detected by Western blot using phospho‐specific antibodies. Results: HIV PIs, atazanavir and lopinavir, activated ERK1/2 and JNK, but not p38. HIV PI‐induced TNF‐¦Áand IL‐6 expression was attenuated by PD98059 and JNKII, but not SB203580. Furthermore, HIV PI‐induced ERK1/2 activation was completely inhibited by CHOP knock down using siRNA or in CHOP knock out macrophages. However, HIV PI‐induced CHOP expression was only inhibited by JNKII but not PD98059 and SB203580. Conclusions: Both ERK1/2 and JNK signaling pathways are involved in HIV PIs‐induced increase of TNF‐¦Á and IL‐6 expression in macrophages. CHOP plays an essential role in HIV PI‐induced inflammatory cytokine release by activating ERK1/2 and JNK signaling pathways in macrophages.
Systemic oxidative stress induced by Diabetes and Residual Oil Fly Ash Inhalation
Authors: Rhoden Claudia Ramos , Chagas Juliana K , Venturini Carina Duarte , Moresco Mauricio , Halmenschlager Graziele , Schmid Helena
Published: June 2021

The importance of evaluating the systemic effects of diabetic subjects exposed to air pollution is notorious, specially to the relevance that both factors have in the world socio‐economic scenario. The aim of the present study is to evaluate the effects of diabetes and chronic exposure to “residual oil fly ash” (ROFA) in the rat systemic oxidative stress. The animals were divided into four groups: diabetic: treated with ROFA (DM POL) − 50μg of ROFA/10μL of saline, intranasal instillation (i.n.); diabetic (DM NPOL) ‐ 10μL of saline, i.n; non‐diabetic treated with ROFA (ND POL) − 50μg of ROFA/10μL of saline, i.n. and non‐diabetic (ND NPOL) ‐ 10μL of saline, i.n., for 60 days. The diabetes was induced by 60mg/kg, i.p. streptozotocin administration. Blood sample was collected on the 30º and 60° days of treatment to evaluate SOD and catalase activity and the lipid peroxidation (through the concentration of malondialdeyde ‐MDA‐). After 30 days, we observed that ND POL animals showed higher levels of SOD comparing to the other groups (p<0.05) while catalase and MDA concentration did not show difference among the groups. After 60 days, the diabetic groups (DM POL and DM NPOL) showed increased levels of MDA. SOD activity was not statistically different among the groups (p> 0.05) while catalase showed statistically increased in the DM POL, DM NPOL and ND POL groups as compared to the control group ND NPOL (p<0.05). The global mortality rate of the DM POL group was 46% while in the other groups this rate was 0%. All deaths occurred after the 30° day of treatment with ROFA. The present study suggests that diabetic condition increases the susceptibility of health adverse effects induced by ROFA instillation and that oxidants could be involved in these effects.

Support: FFFCMPA
An aqueous extract of Zanthoxylum schinifolium has a positive inotropic effect in beating rabbit atria
Authors: Cui Hao Zhen , Kang Dae Gill , Kim Seung Ju , Choi Deok Ho , Cho Kyung Woo , Lee Ho Sub
Published: June 2021

The positive inotropic effect of the aqueous extract of Zanthoxylum schinifolium (AZS) and the possible mechanisms responsible for this effect were investigated in beating rabbit atria. AZS significantly increased atrial stroke volume, pulse pressure, and cAMP efflux, but decreased ANP secretion in beating rabbit atria. These effects were restricted by pre‐treatment with diltiazem or veraparmil, L‐type Ca2+ channel blockers, and staurosporine, a non‐selective protein kinase inhibitor, respectively. The effects of AZS on atrial dynamics were also abolished by pre‐treatment with KT‐5720, a protein kinase A inhibitor, but was not altered by treatment of chelerythrine, a protein kinase C inhibitor. Treatment of atrial membrane with AZS obviously increased the cAMP production induced by forskolin, but not increased by 3‐isobutyl‐1‐methylxanthine (IBMX). Furthermore, co‐treatment with AZS and Ro 20‐1724, a phosphodiesterase (PDE)‐4 inhibitor, more produced atrial cAMP than Ro 20‐1724 alone. However, there were no increase in atrial cAMP production by co‐treatment with AZS and milrinone, a PDE‐3 inhibitor, compared with milrinone alone. These results suggest that the AZS‐induced positive inotropic activity may be due to the enhancement of cAMP level by PDE‐3 inhibition in beating rabbit atria.
Studies of de novo Purine Biosynthetic Complexes in Living Cells
Authors: Benkovic Stephen J , An Songon , Kumar Ravindra , Sheets Erin D
Published: June 2021

Purines are synthesized in ten chemical steps catalyzed by six enzymes in eukaryotes. Studies in vitro have provided scant evidence for anticipated protein‐protein interactions that would enable substrate channeling and regulation of the metabolic flux. We have applied fluorescence microscopy to HeLa and human breast cancer cells and discovered that all six enzymes colocalize to form clusters in the cellular cytoplasm. The association and dissociation of these enzyme clusters can be regulated dynamically by changing the purine levels or by the addition of exogenous agents to the culture media. Collectively, the data provide strong evidence for the constitution of a multi‐enzyme complex, the purinosome, to carry out de novo purine biosynthesis in cells.
Norepinephrine (NE) Modulates Active Caspase‐3 Expression and Increase Susceptibility of Cardiomyocytes for Voltage‐stimulated Deformation during Sepsis
Authors: Chopra Mani , Das Padmalaya , Sharma Avadhesh C.
Published: June 2021

In the present study, we tested the hypothesis that the progression of sepsis from 1 to 7‐days modulates the NE‐induced alterations in caspase‐3 and the time for deformation (TDEF) of adult rat ventricular myocytes (ARVM). Male Sprague‐Dawley rats (350–400g) were randomized into sham, 1‐, 3‐ and 7‐day sepsis groups. Sepsis and sham‐sepsis was induced using 200 mg/kg cecal inoculum and 5% dextrose water i.p., respectively. Isolated ARVMs were subjected to 5Hz stimulation using the IonOptix edge detection system to record TDEF (cutoff time 300s). The concentration of myocardial NE and the expression of active caspase‐3 were determined using immunochemical assay in the myocardial tissue samples. The cell viability of the NE‐treated septic ARVMs was significantly reduced (42%) compared to (26%) in the sham NE‐treated ARVMs. When stimulated, the septic ARVMs produced a progressive decrease in TDEF in the 1‐day (195±38s), 3‐day (156±22s) and 7‐day (101±28s) groups compared to the sham (206±28s) group. The NE‐treated septic ARVMs significantly decreased TDEF in both the sham and septic ARVMs. A significant increase in the myocardial NE levels was observed in the septic groups. We observed that the increased duration of sepsis significantly elevated the myocardial active caspase‐3, which was further upregulated by NE. The data suggest that an NE‐induced decrease in the TDEF correlated with the upregulated expression of active caspase‐3. We concluded that active caspase‐3 may increase the susceptibility of NE‐treated ARVMs for voltage‐stimulated deformation during progression of polymicrobial sepsis. (This work was supported by NHLBI # 66016).
Variability in valproic acid (VPA) metabolite signatures in children
Authors: Leeder James Steven , Gaedigk Andrea , Pearce Robin E. , Abbott Frank S. , Chang Thomas K.H. , Kiang Tony , Teng XiaoWei , Kennedy Mary Jayne , Ward Robert M.
Published: June 2021

VPA is associated with an idiosyncratic hepatotoxicity that is most prevalent in children under 2 years of age receiving concurrent enzyme‐inducing anti‐epileptic drugs. The goal of this study was to establish the limits of normal variability in VPA metabolite patterns in a population of children. Urine was collected over a steady state dosing interval in 91 pediatric patients aged 2 to 17 years receiving VPA either as monotherapy or polytherapy, and analyzed by GC‐MS methods for VPA and 14 metabolites. Principal components analysis of the log‐transformed metabolite data (corrected for urinary creatinine) revealed three distinct clusters of metabolites generally corresponding to microsomal biotransformation, mitochondrial beta‐oxidation, and N‐acetylcysteine (NAC) metabolites derived from glutathione (GSH)‐conjugates of (E)‐2,4‐diene. If NAC conjugates are the products of reactive metabolite formation and GSH conjugation in mitochondria and free 2,4‐diene‐VPA is derived from a non‐reactive microsomal pathway, we hypothesize that the NAC‐VPA/2,4‐diene ratio may reflect interindividual variability in bioactivation and detoxification capacity. A Q‐Q plot of the log‐transformed ratio implies a bimodal distribution that is being evaluated as a potential biomarker for VPA‐toxicity in this population.

Supported by grant U01 HD‐044239 from NICHD
Expression and functional characterization of murine organic anion transporting polypeptide 1b2 (oatp1b2/oatp4/lst‐1)
Authors: Meyer zu Schwabedissen Henriette E. , Ware Joseph A. , Tirona Rommel G. , Kim Richard B.
Published: June 2021

The organic anion transporting polypeptide family plays important roles in mediating the cellular uptake of endogenous and exogenous compounds. Human liver enriched transporters such as OATP1B1 and OATP1B3 have been widely studied and shown to be involved in hepatic uptake and disposition of hormones, bile acids, and drugs. However, little is known about the murine orthologue, oatp1b2. Accordingly, expression of mouse oatp1b2 was assessed using real‐time PCR and Western blot analysis. mRNA transcripts were detected in a number of tissues including kidney, skeletal muscle, stomach and heart, with highest level in mouse liver. Immunohistochemical detection revealed localization of oatp1b2 in the sinusoidal membrane of hepatocytes. In contrast to the mRNA data, oatp1b2 protein expression was not detected in other tissues but liver. Cloning of the full coding region of oatp1b2 revealed the presence of two new splice variants. Interestingly, these variants were significantly expressed in the organs noted previously, but much less in liver. Heterologous expression experiments revealed taurocholic acid, estrone‐3‐sulfate, estradiol‐17β‐glucuronide, pravastatin and cerivastatin as substrates of the transporter. These findings give insights to species related differences in OATP transporter function that has application to mouse models of hepatic drug disposition.
Functional Characterization of Polymorphic Variants in the Human Apical Sodium‐dependent Bile Acid Transporter (ASBT; SLC10A2)
Authors: Ho Richard H. , Leake Brenda F. , Dawson Paul A. , Kim Richard B.
Published: June 2021

The apical sodium‐dependent bile acid transporter (ASBT) mediates the uptake of bile acids into intestinal enterocytes, supporting a critical role in normal bile acid homeostasis and maintenance of enterohepatic recirculation. Complete loss of function mutations in ASBT are associated with primary bile acid malaborption, characterized by diarrhea, steatorrhea, interruption of bile acid enterohepatic circulation, and reduced plasma cholesterol levels. We identified 6 nonsynonymous polymorphic variants within the exonic regions of the SLC10A2 gene from DNA derived from a large number of ethnically diverse healthy subjects. In general, variants tended to be infrequent and ethnic‐dependent; only one variant, 511GT (Ala171Ser), was found with a relatively common allele frequency (6.0%), in European‐Americans. A recombinant vaccinia‐based system was utilized to heterologously express wild‐type and variant ASBT variants in HeLa cells for functional studies using radiolabeled taurocholate as a substrate. Two variants, 292GA (Val98Ile) and 431GA (Cys144Tyr), were associated with significantly impaired taurocholate transport activity in vitro compared to wild‐type ASBT. Accordingly, polymorphisms in SLC10A2 may have important functional implications to normal bile acid homeostasis and enterohepatic recirculation. This work was supported in part by NIH grants GM54724 (RBK) and GM81363 (RHH).
Green tea catechin EGCG suppresses NUDT6 (nudix [nucleoside diphosphate linked moiety X]‐type motif 6) expression in human colorectal cancer
Authors: Sukhthankar Mugdha Girish , Cekanova Maria , Baek Seung Joon
Published: June 2021

A natural antisense RNA of FGF‐2, FGF‐ AS, has been implicated to modulate the function of pro‐angiogenic factor FGF‐2 (bFGF). FGF‐AS RNA encodes a novel nudix motif protein, NUDT6 /GFG, whose function is not yet known. The main aim of this study was to characterize the transcriptional regulation and biological function of this protein. Our microarray data showed that a green tea component, epigallocatechin (ECG), suppressed NUDT6 expression, and this was confirmed by RT‐PCR. Subsequently, the use of different catechins showed that the effect of epigallocatechin gallate (EGCG) was stronger than that of ECG. At the post‐transcriptional level, EGCG affected the RNA stability of NUDT6, indicating it as a potential mechanism of NUDT6 suppression. To further define the underlying mechanism of biological function of NUDT6 in human colorectal cancer cells, we constructed the NUDT6 correct (pool) and reverse (control) orientation clones, tagged with V5. These plasmids were transfected and selected under G418 in HCT‐116 cells to generate stable cell lines for control and NUDT6. Using these cell systems, we found that NUDT6 expression increased cell growth, soft agar cloning efficiency, and colony formation, compared to the control cells. There was also decreased 3/7‐caspase activity in pool compared to control. We conclude that NUDT6 is a novel cell proliferator in colorectal cancer and is down‐regulated by the green tea catechin EGCG.
Inhibition of the Nrf2‐Antioxidant Pathway Sensitizes Cells to Alkylating Anticancer Drugs
Authors: Kwak Mi‐Kyoung , Cho Jeong‐Min , Manandhar Sarala , Lee Hyang‐Rim
Published: June 2021

The treatment of alkylating cytotoxic drug cisplatin is often limited by high incidence rate of resistance. In the present study, the potential involvement of the transcription factor Nrf2 in determination of cisplatin cytotoxicity has been investigated. Nrf2‐deficient murine embryonic fibroblasts showed increased cell death, cytotoxicity, and apoptosis in response to cisplatin treatment compared to wild‐type cells. Cisplatin‐resistant human ovarian cancer SK‐OV cells, which are retaining 25‐fold higher levels of GSH than murine fibroblasts, could be sensitized by inhibition of Nrf2. Transfection with Nrf2 siRNA into SK‐OV cells resulted in severe degree of GSH depletion and exacerbated cytotoxicity following cisplatin treatment compared to scrambled RNA control. In conclusion, we propose that the Nrf2 pathway, which plays a protective role in normal cells, can be a potential target to control cancer cell resistance to oxidants, cytotoxic chemicals, and radiation.
Combined use of Ganoderma lucidum ReishiMax and tea polyphenols Tegreen inhibits tumor growth synergistically in cancer‐bearing mice
Authors: Zhang Yan , Zhang Li , Tan Ningzhi , Qi Ying , Gao Ling , Zhu Jia‐Shi
Published: June 2021

Ganoderma lucidum (Reishi) and green tea are commonly used in China for cancer prevention and adjuvant therapy. A screening revealed superior anti‐malignancy activities of ReishiMax (RM) among commercial Reishi products (JACM 9:491, 2003). Synergy of RM and Tegreen (TG; containing >98% tea polyphenols) was found in anti‐cancer in human breast and prostate cancer cells (Itnl J Oncol 30:963, 2007). Further screening showed that fucose‐containing polysaccharides are highly enriched in RM (0.34%), the class of polysaccharides demonstrating high immune enhancing activities (Bioorg Med Chem 10:1050, 2002). They are much less (0.04–0.20%) in other Reishi products. In addition to the survival studies in cancer mice (FASEB J 21:A1100, 2007), we tested synergy of RM and TG in inhibiting tumor growth in sarcoma‐bearing mice. After 28‐day treatment, RM+TG significantly decreased tumor weight by 45% in S180‐innoculated mice, compared to controls (p<0.05). The data further confirmed the previous finding of synergy of RM and TG in anti‐malignancy, indicating their therapeutic values in cancer prevention and adjuvant treatment.
Cannabinoid receptor‐1 deletion or inhibition protects against doxorubicin‐induced cardiotoxicity.
Authors: Pacher Pal , Mukhopadhyay Partha , Bátkai Sandor , Rajesh Mohanraj , Haskó György , Kunos George
Published: June 2021

Doxorubicin(DOX) is a potent antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Endocannabinoids mediate cardiodepressive effects through cannabinoid 1 (CB1) receptors in various pathophysiological conditions, and these effects can be reversed by CB1 antagonists. We aimed to explore the effects of genetic deletion or pharmacologic inhibition of CB1 receptor in in vivo and in vitro models of DOX‐induced cardiotoxicity. Five days after the administration of a single dose of DOX (20 mg/kg intraperitoneally) to mice various load‐dependent/independent indexes of contractility were significantly depressed, and the myocardial level of the endocannabinoid anandamide was elevated compared with vehicle‐treated control mice. Genetic deletion of CB1 receptor or its pharmacological inhibition markedly improved cardiac dysfunction and reduced DOX‐induced apoptosis in the myocardium. Doxorubicin also decreased cell viability and induced apoptosis in the H9c2 myocardial cell line, which were prevented by the preincubation of the cells with CB1 antagonist, which also beneficially modulated various key signaling processes involved in the detrimental effects of DOX. These data suggest that CB1 antagonists may represent a new cardioprotective strategy against DOX‐induced cardiotoxicity.
Glutamate supplementation prevents Helicobacter pylori‐induced gastric mucosal damages in gerbils
Authors: Nakamura Eiji , Amagase Kikuko , Hasumura Mai , Gabriel Ana San , Uneyama Hisayuki , Torii Kunio , Takeuchi Koji
Published: June 2021

Backgrounds & Aims: Chronic Helicobacter pylori (HP) infection induces gastric diseases such as gastritis, gastric ulcers and gastric cancers. The importance of urease‐derived ammonia in the pathogenesis of HP‐induced gastric diseases is known by the fact that Helicobacter felis (expresses urease but not vac A or cag PAI‐related gene products) induces gastritis and gastric cancer in animal models. Glutamate is previously reported to protect cultured gastric epithelial cells against ammonia (Am J Physiol 2002). However, whether glutamate protects in vivo gastric mucosa against HP is still unknown. Thus, we examined the effect of glutamate supplementation on HPi‐induced gastric mucosal damage using gerbil model.

Materials & Methods: The diets with or without glutamate supplementation were fed to HPi‐infected Mongolian gerbils for 3 months, then the gastric mucosa were used for the macroscopic, histochemical analyses. The number of viable HP in the stomach was also determined.

Results & Discussion: Glutamate supplementation significantly suppressed HP‐induced gastric mucosal damage and inflammatory cell infiltration. Interestingly, the number of viable HP in the stomach was unchanged between the diet with and without glutamate.

Conclusion: Glutamate supplementation protects HP‐induced gastric mucosal damage without affecting HP itself, suggesting the stimulatory role of glutamate on gastric defensive factors.
Regulation of VCAM‐1 expression by TNF‐α in cerebral arteries of control and diabetic mice
Authors: Nilsson‐Öhman Jenny , Nilsson Lisa M , Agardh Carl‐David , Agardh Elisabet , Gomez Maria F
Published: June 2021

Diabetic patients suffer from micro‐ and macroangiopathy. Previous studies show that plasma levels of the pro‐inflammatory cytokine TNF‐α are elevated in diabetic patients and that this cytokine promotes atherosclerosis, but the role of TNF‐α in the development of diabetic microangiopathy is unclear. Upregulation of the cell adhesion molecule VCAM‐1 is one of the earliest detectable responses to vascular disease. Here we investigate the impact of TNF‐α signaling on VCAM‐1 expression in small resistance arteries (10 to 250 μm) from WT, Apoe−/−, TNF‐α−/− and Apoe−/−/TNF‐α−/− mice, under normal or diabetic conditions. Using confocal immunofluorescence microscopy, we show that VCAM‐1 is expressed in both endothelial and smooth muscle cells and that higher expression levels correlate to larger caliber vessels. Loss of TNF‐α resulted in decreased VCAM‐1 expression, whereas loss of TNF‐α on an Apoe−/− background had no effect. Contrary to expectations, arteries from Apoe−/− did not show higher VCAM‐1 levels than those of WT mice. Interestingly, only arteries from mice lacking TNF‐α responded with increased VCAM‐1 expression when the animals became diabetic. These results suggest that TNF‐α may regulate VCAM‐1 expression in small arteries under basal conditions, but not under diabetic conditions. Supported by: Swedish Heart & Lung Foundations, Research Medical Council & Royal Physiographic Society in Lund