bluuebunny/crossref_metadata_2025 huggingface dataset:
Decreased Ovalbumin‐induced Airway Inflammation in Mice Deficient in the Sensory Neuronal Ion Channel TRPA1
Authors: Caceres Ana Isabel , Brackmann Marian , Bessac Bret F , Cohn Lauren , Jordt Sven Eric
Published: June 2021

The airways are densely innervated by peripheral sensory neurons. Activation of chemosensory C‐fibers by chemical irritants triggers defensive reflexes such as cough and sneezing, and initiates airway constriction and glandular fluid secretion. Local neuronal release of pro‐inflammatory peptides promotes edema formation and vascular leakage. We recently identified TRPA1 as the chemosensory receptor for noxious oxidants and electrophiles in the airways.

Chemosensory neurons are activated or sensitized during chronic inflammatory states and may be involved in the initiation and maintenance of airway conditions such as asthma and chronic obstructive pulmonary disease (COPD). Inflammatory mediators, including prostaglandins, bradykinin, histamine, reactive oxygen species and lipid peroxidation products activate or sensitize TRPA1 and other sensory ion channels such as TRPV1, the capsaicin receptor.

We asked whether absence of TRPA1 or TRPV1 would affect the inflammatory process in the ovalbumin (OVA) mouse model of allergic airway inflammation. OVA‐challenged TRPA1‐deficient mice showed dramatically diminished eosinophil infiltration in the airways. Multiplex analysis of bronchoalveolar lavage fluid showed a reduction in levels of Th2‐derived cytokines and chemokines.

We conclude that neuronal TRPA1 channels are essential for establishment of OVA‐dependent airway inflammation in mice.
The effect of morphine on a K+ channel from a murine enteric neuron cell line derived from the H‐2kb‐tsA58 mouse
Authors: Hawkins Gregory , Dewey William , Srinivasan Shanthi , Akbarali Hamid
Published: June 2021

Historically the generation and study of isolated primary murine enteric neurons (EN) has proven extremely difficult. Recently an EN cell line has been developed from the H‐2kb‐tsA58 mouse (immortomouse). In the present study we examined ion channel expression in these ENs. Single channel recordings were made by cell attached patch clamp with high K+ (140mM) in the bath and KCl (140mM), HEPES (10mM) in the pipette in order to zero the transmembrane potential. We have identified an outward current with average amplitude of 3.8 pA at +40mV. The conductance of this channel was 10.2 pS and was blocked by 130mM Cs+ in the pipette solution. Application of 1 µM morphine in the bath decreased the open probability of this channel from 0.01945 to 0.00634. Expression of µ‐opioid receptors were detected by immunocytochemistry. Our findings demonstrate that EN cells express a K+ channel that is sensitive to morphine and which may play an important role in the study of opioid bowel disorders.
Modulation of Myocardial Mitochondrial VDAC/ MitoKATP Channels in Sepsis
Authors: Das Padmalaya , Chopra Mani , Sharma Avadhesh C
Published: June 2021

In the present study, we tested the hypothesis that norepinephrine by modulating myocardial voltage‐dependent anion channel (VDAC)/mitoKATP channels can affect the release of cytochrome C and regulate intrinsic apoptotic cascade. Male Sprague‐Dawley rats (350‐400g) were made septic using 200mg/kg cecal inoculum, ip. Sham animals received 5% dextrose water, ip. VDAC mRNA and protein expression was determined in the myocardium and isolated single adult rat ventricular myocytes (ARVMs) obtained from sham and septic rats. In addition, mitochondria and cytosolic fractions were isolated from ARVM treated with norepinephrine (NE, 10ìmoles) in the presence and absence of 5‐Hydroxydecanoic acid (5HD; 100ìmoles), a selective mitoKATP channel blocker. Sepsis produced a significant down‐regulation of VDAC mRNA and protein along with upregulation of Bax in both myocardium and ARVMs. Pretreatment of septic ARVMs with 5HD blocked NE‐induced decrease in VDAC expression and the release of cytochrome C. These results suggest that sepsis downregulates myocardial VDAC expression. It appears that NE modulates mitoKATP channels and increases the release of cytochrome C, which may play a crucial role in sepsis‐induced cardiomyocyte dysfunction.
Ubiquitous inactivation of TRPM4 leads to elevated blood pressure in mice
Authors: Mathar Ilka , Vennekens Rudi , Meissner Marcel , Flockerzi Veit , Schweda Frank , Freichel Marc
Published: June 2021

Recently we showed that transient receptor potential TRPM4 proteins act as Ca2+‐activated nonselective cation channels in mast cells that critically limit the driving force for Ca2+ influx, release of inflammatory mediators and anaphylactic responses. In addition to mast cells we detected TRPM4 proteins in heart, aortic endothelial cells, kidney and adrenal gland from mice. Earley et al suggested that TRPM4 is expressed in vascular smooth muscle cells and it is supposed to act as a mechanosensor regulating vascular myogenic tone.

Telemetric blood pressure measurements reveal that mean arterial blood pressure (MAP) is elevated in TRPM4‐/‐ mice by 11mmHg under basal conditions. Blood pressure elevation is observed during the complete circadian period and most notable under resting conditions. Plasma aldosterone levels, renin mRNA expression in the kidney and renin plasma concentrations are not different in TRPM4‐/‐ mice and there are no differences in MAP in response to application of phenylephrine (PE), norepinephrine and L‐NAME. PE induced contraction and acetylcholine induced relaxation of aortic rings is not altered. Currently we analyse the contractility of resistance vessels with respect to smooth muscle‐dependent contraction and endothelium‐dependent relaxation. Inhibition of ganglionic transmission with hexamethonium abolishes the difference in MAP between both genotypes similarly like application of prazosin. Analysis of plasma catecholamine level revealed a significant elevation of epinephrine in TRPM4‐/‐ mice. These results suggest that an increased neurogenic tone contributes to the development of the hypertension.
The AMPK activation by sauchinone, a Saururus chinensis lignan, enables hepatocytes to protect against the toxicity induced by iron overload
Authors: Shin Sang Mi , Kim Young Woo , Kim Sang Geon
Published: June 2021

Iron overload disorder may lead to liver fibrosis and hepatocellular carcinoma. Sauchinone, a lignan in Saururus chinensis, inhibits the induction of iNOS and TNF‐alpha in macrophages. This study investigated the hepatoprotective effect of sauchinone in mice challenged with phenylhydrazine (PHZ), an in vivo model representing hepatic iron overload. PHZ treatments that elevated iron and arachidonic acid (AA, a proinflammatory lipid) levels in plasma caused liver injury, which was attenuated by concomitant sauchinone treatments. The molecular basis of sauchinone′s hepatoprotective effect was explored in a hepatocyte model. Combination treatment of iron and AA synergistically increased cytotoxicity, as supported by increases in reactive oxygen species, mitochondrial dysfunction and apoptosis, all of which were prevented by sauchinone. Sauchinone activated AMPK via LKB1, which was responsible for its cytoprotective effect, as shown by an experiment using a dominant negative mutant construct. AMPK activation by sauchinone was confirmed in the mouse liver. These results demonstrated that sauchinone enables hepatocytes to protect against iron‐induced toxicity, which might be associated with its AMPK activation.
Endothelial Focal Adhesion Kinase Depletion Augments Lung Vascular Permeability by Impairing Sphingosine‐1‐Phosphate Receptor‐1 Function
Authors: Thennes Tracy , Knezevic Nebojsa , Tauseef Mohammad , Yla‐Herttuala Seppo , Shen Tang‐Long , Guan Jun‐Lin , Mehta Dolly
Published: June 2021

We have shown that focal adhesion kinase (FAK), a determinant of cell‐matrix adhesion, maintains endothelial barrier function and limits the increase in endothelial permeability induced by inflammatory mediators. We conditionally depleted endothelial FAK (CD‐FAK) using Tie2‐Cre in FAK‐floxed mice to investigate the specific role of FAK in regulating lung vascular permeability (LVP). LVP was determined by measuring lung wet/dry ratio and Evans blue albumin accumulation in lung parenchyma. We show CD‐FAK mice have increased basal LVP, and activation of thrombin receptor PAR1 further augmented this increase. Sphingosine‐1‐phosphate (S1P), by binding to S1P receptor‐1 (S1P1), strengthens the endothelial barrier via Rac1 mediated adherens junction (AJ) assembly. We assessed the effect of FAK knockdown in S1P1 signaling. Depletion of FAK prevented S1P1‐mediated Rac1 activation, AJ assembly, and endothelial barrier strengthening. S1P could only partially reverse PAR1‐induced increase in LVP in CD‐FAK mice. Since S1P1 contains tyrosine residues, we examined if FAK regulated S1P1 signaling by post‐translationally modifying S1P1. We show that S1P stimulated the interaction between S1P1 and FAK, and that S1P induced tyrosine phosphorylation of S1P1 in a FAK‐dependent manner. These findings identify S1P1 as a novel effector of FAK, which upon activation by FAK, may serve as a mechanism to limit basal LVP.
Identification of the 14‐3‐3/HIP‐55 protein complex as a negative regulator of HPK1 in the Akt signaling
Authors: Li Zijian , Shi Zhi , Park Hae Ryoun , Li Zenggang , Fu Haian
Published: June 2021

HIP‐55 (hematopoietic progenitor kinase 1 [HPK1]‐interacting protein of 55 kDa) is a multidomain protein that contains an actin‐binding domain at its N terminus and an SH3 domain at its C terminus. It functions in part through controlling the HPK1 function, which appears to be important for organ development and immune response. HIP‐55 deﬁciency caused abnormal structure and function of multiple organs, including the spleen, heart, and lung in both heterozygous and homozygous mice. Here report the identification of a novel HIP‐55 partner protein, 14‐3‐3, with a proteomic approach from lung epithelial cells. We found that the HIP‐55/14‐3‐3 interaction is controlled by Akt. Mutational analysis revealed Ser‐269 and Thr‐191 as critical sites for Akt phosphorylation, which are essential for mediating 14‐3‐3 binding. While HIP‐55WT(wide‐type) decreased the kinase activity of HPK1, mutant HIP‐55 that is defective in Akt phosphorylation and 14‐3‐3 binding failed to inhibit HPK1. This study couples the HIP‐55/HPK1 regulatory axis to an Akt‐mediated signaling pathway and identifies a novel mechanism by which HIP‐55 suppresses HPK1 in an Akt/14‐3‐3‐dependent manner. Our findings reveal a novel Akt‐regulated signaling mechanism that controls HPK1 mediated functions, such as organ development, immune response, and tumorigenesis.

This work was supported by Emory Global Health Institute and NIH PO1 CA116676.
Angiotensin II Induces JAK2 and IL‐6 in Brainstem Rat Astrocytes
Authors: KANDALAM UMADEVI , CLARK MICHELLE A
Published: June 2021

Tyrosine kinases and mitogen‐activated protein kinases are known mediators of angiotensin II (Ang II) effects in cultured rat astrocytes. In this study, we investigated whether Ang II induced Janus kinase (JAK) 2 phosphorylation and interleukin‐6 (IL‐6) secretion in brainstem rat astrocytes. Ang II increased JAK2 phosphorylation in a time‐ and concentration‐dependent manner. Maximal effect was observed at 15 min with 100 nM Ang II. Losartan (10 µM), an AT1 receptor antagonist, inhibited Ang II‐mediated JAK2 phosphorylation, while 10 μM PD123319, an AT2 receptor blocker, was ineffective. The JAK2 inhibitor, AG490 (50 µM) prevented Ang II JAK2 phosophorylation. Ang II (100 nM) also stimulated IL‐6 secretion from brainstem astrocytes with maximal secretion at 48 hrs. Losartan decreased Ang II‐induced IL‐6 secretion while PD123319 was ineffective. Interestingly, AG490 reduced Ang II‐stimulated IL‐6 secretion. These findings showed for the first time that Ang II induced JAK2 phosphorylation and IL‐6 secretion through activation of the Ang II AT1 receptor in brainstem astrocytes. In addition, Ang II stimulated IL‐6 secretion through the JAK‐2 pathway in brainstem astrocytes. These results provide new insights into Ang II signaling in astrocytes. (Support: NHLBL HLO77199 and by Nova Southeastern University President's Faculty Research & Development Grant.).
Identification of a Novel Regulatory Element Controlling ASK1‐JNK Signaling
Authors: Puckett Mary Clark , Cockrell Lisa M , DeSimone Mia , Vellano Chris , Fu Haian
Published: June 2021

Jun N‐terminal kinase (JNK) plays a key role in mediating diverse signaling events and controls a number of important cellular processes such as apoptosis, proliferation, metabolism, and DNA repair. Dysregulated activation of JNK signaling has been implicated in numerous disease states, including neurodegenerative diseases, chronic inflammatory disease, ischemia, diabetes, and cancer. Thus, understanding how JNK is regulated may lead to the development of novel therapeutic strategies. Our research focuses on the ASK1 signalosome, an upstream kinase complex that controls JNK activity. Here we report the identification of a novel regulatory element between ASK1 and its associated protein kinase, ASK2. Using protein complex pull‐down assays coupled with mutagenesis, we have conclusively established a ternary protein complex composed of ASK1, ASK2, and 14‐3‐3. The ASK1/ASK2 interaction is essential for 14‐3‐3 association. Through deletion mapping and binding assays, we have identified a critical sequence in ASK2 needed for the ASK1/ASK2 interaction. Expression of this regulatory sequence disrupted the ASK2/ASK1 interaction and inhibited JNK activation in response to stress signals, including hydrogen peroxide. These data established the importance of ASK2 in the ASK1 signalosome for JNK regulation and revealed a regulatory element essential for the control of ASK1‐JNK signaling. The ASK1/ASK2 interface may offer a therapeutic target for manipulating JNK signaling. The identified ASK2 sequence may serve as a basis for the development of novel JNK signaling modulators.

The work was supported by NIH P01 CA116676.
Caveats of proteomics approaches in identifying novel spinophilin interacting proteins
Authors: Baucum Anthony Jerome , Ham Amy‐Joan Lorna , Colbran Roger James
Published: June 2021

Spinophilin is an F‐actin and protein phosphatase 1 (PP1) binding protein that targets PP1 to the postsynaptic density and to various dendritic substrates. PP1 binding to spinophilin is enhanced in an animal model of parkinsonism. Several other known spinophilin associated proteins (SpAPs) were not detected in spinophilin complexes isolated from rat or mouse striatum. Therefore, a de novo shotgun proteomics strategy was developed to identify SpAps. Complexes specifically isolated using goat, mouse or rabbit spinophilin antibodies contained spinophilin, PP1, and neurabin (a known SpAP), as well as novel candidate SpAPs. We selected one of the novel proteins for further validation: the thousand and one amino acid kinase (TAO1). While TAO1 and spinophilin were detected in the reciprocal precipitations, and the amount of co‐immunoprecipitation was reduced following dopamine depletion, TAO1 was also present in spinophilin immunoprecipitates isolated from extracts of spinophilin knockout (KO) mice. Therefore, to identify spinophilin‐selective interactions, spinophilin was immunoprecipitated from both WT and KO animals, using multiple antibodies, and these samples were analyzed by shotgun proteomics. Multiple proteins were present in complexes isolated from WT, but not KO, tissue and studies are ongoing to validate these interactions.
Disruption of the phosphodiesterase 8B (PDE8B) gene alters the hypothalamic‐pituitary‐adrenal (HPA) axis
Authors: Tsai Li‐Chun Lisa , Shimizu‐Albergine Masami , Beavo Joseph A.
Published: June 2021

Corticosterone, the major murine glucocorticoid, is synthesized and secreted by adrenal zona fasciculata cells in response to the pituitary hormone adrenocorticotropin (ACTH) in a cAMP/protein kinase A dependent manner. Circulating corticosterone then provides negative feedback to the HPA axis forming an efficient regulatory loop that maintains hormone homeostasis. Important physiological functions of corticosterone include regulation of glucose metabolism and inflammation. Intracellular levels of cAMP are controlled through regulation of its synthesis by adenylyl cyclases and degradation by PDEs. We previously reported that the cAMP specific PDE8B is highly enriched in adrenal fasciculata cells. We also observed that PDE8B knockout mice have elevated urinary corticosterone. Here we report that cultures of primary adrenocortical cells isolated from PDE8B knockout mice show increase responsiveness to ACTH compared to paired control mice. We also observe changes in circulating ACTH levels and mRNA levels of some steroidogenic enzymes in PDE8B knockout mice compared to controls. These findings suggest that PDE8B plays an important role in the regulation of adrenal steroidogenesis and that the absence of PDE8B could cause adrenal hypersensitivity in vivo. Supported by NIH grants DK21723 and GM083926.
Phosphodiesterase 8A Modulates Lipolysis in Primary Brown Adipocytes
Authors: Kraynik Stephen , Patrucco Enrico , Shimizu Masami , Beavo Joseph
Published: June 2021

Brown adipose tissue (BAT) is the primary center of metabolic heat production in mammals during cold‐induced, adaptive thermogenesis. While white fat is an energy "storing" tissue, BAT is known as an energy "wasting" tissue, where the energy from triglyceride breakdown is lost as heat. Intracellular increases in cAMP are known to regulate a number of key processes in BAT, such as acute lipolysis, precursor proliferation and differentiation, and the induction of thermogenic genes including Ucp1. Phosphodiesterases (PDE) are enzymes that catalyze the breakdown of cyclic nucleotides, thereby modulating the second messenger's downstream effects. Here, we present evidence that Phosphodiesterase 8A (PDE8A) is expressed highly in the interscapular BAT of mice, though not expressed in white fat. Utilizing a PDE8A‐/‐ mouse, which has a LacZ‐Neo cassette inserted into the endogenous PDE8A locus, we observe strong β‐Gal activity in BAT. We confirmed PDE8A expression in wild‐type BAT using mRNA, protein, and activity analyses. Preliminary evidence shows that PDE8A ablation modulates lipolysis in response to isoproterenol in isolated adipocytes, suggesting that PDE8A plays a role in the lypolitic component of the thermogenic response in brown fat. Supported by NIH grants DK21723 and GM08392.
Different G protein γ subunits co‐precipitate with the adenosine A1 and A2A receptor subtypes
Authors: McIntire William , Bigler‐Wang Dora , Figler Heidi , Figler Robert , Linden Joel , Garrison James
Published: June 2021

G protein βγ dimers are a critical component in the transfer of information from activated receptor to G protein, as well as regulators of downstream signaling. An unresolved question is how a receptor interacts with the large complement of β and γ isoforms within a given cell. Identity of β subunits precipitated with receptor can be readily determined with isoform specific antibodies, whereas characterization of γ isoforms has proven more difficult. To address this, expression vectors containing epitope tagged adenosine A1 or A2A receptors were developed to express these receptors in mammalian cell lines. Cell membranes were solubilized in 1% digitonin, the receptor:G protein complexes purified by FLAG affinity chromatography and separated by SDS‐PAGE. The Gγ containing gel band was excised, digested with trypsin, and the resulting peptides extracted, separated by HPLC and sequenced by mass spectrometry. In HEK‐293 cells, γ5, γ7, γ10 and γ12 isoforms were found to be associated with both Gi coupled adenosine A1 and Gs coupled adenosine A2A receptors; however, the γ2 and γ4 isoforms were observed with the adenosine A2A receptor, but not the A1 receptor, suggesting that the different receptors couple to different γ isoforms. This research supported by NIH grant DK‐19952 and AHA grant 0535350N.
Gi G‐protein Selective RGS Proteins Regulate Lysophosphatidic Acid Signaling in SKOV‐3 Ovarian Cancer Cells
Authors: Hurst Jillian H. , Callihan Phillip , Turner Kathryn , Hooks Shelley B.
Published: June 2021

Lysophosphatidic acid (LPA) drives ovarian cancer progression by activating multiple cell surface receptors coupled to heterotrimeric G‐proteins. Regulator of G‐protein Signaling (RGS) proteins accelerate the deactivation of G‐proteins, suggesting that they may negatively regulate LPA signaling in ovarian cancer cells (OCCs). We have previously reported that migration, growth, and MAP kinase activation are all Gi‐dependent processes in SKOV‐3 OCCs. Further, SKOV‐3 cells expressing RGS‐insensitive Gαi2 subunits have greater responses to LPA in assays measuring inhibition of adenylyl cyclase, migration, growth, and MAP kinase activation compared to cells expressing wild‐type Gαi2. Based on comparative transcript expression data between normal and transformed ovarian cell lines and normal and tumor clinical samples, several differentially expressed RGS transcripts have been identified and we have selected several Gi‐targeted RGS proteins expressed in SKOV‐3 cells for functional studies. LPA‐stimulated cell growth and migration were evaluated in cells following RGS overexpression via plasmid transfection or RGS knock‐down following transfection of synthetic siRNAs. Using this approach, the effect of specific RGS proteins on LPA signaling in ovarian cancer cells is being defined. This research is supported by the Elsa U. Pardee Foundation and the Georgia Cancer Coalition.
Regulation of AGS3 and Gialpha1 interaction in living cells
Authors: Oner Sukru Sadik , Ningfei An , Breton Billy , Bouvier Michel , Blumer Joe , Stephen Lanier
Published: June 2021

Activator of G Protein Signaling 3 (AGS3) and related GPR proteins provide unexpected regulatory mechanisms for G‐protein signaling systems. AGS3 contains 4 GPR motifs, each of which can serve as a docking site for Giα‐GDP free of Gβγ. Two of the key questions for AGS3 is what controls its interaction with G‐protein and where does this interaction occur within the cell. As an initial approach to this question, we evaluated the interaction of AGS3 with Giα1 in living cells using bioluminescence resonance energy transfer (BRET) to measure interaction between proteins tagged with Renilla luciferase (RLuc) and the YFP‐variant Venus in HEK cells. AGS3 and Giα1 reciprocally tagged with either RLuc or YFP exhibited a BRET signal that was saturable and specific. The net BRET signal was blocked by Gβγ expression and it was not observed with the AGS3‐Q/A mutant in which each of the GPR motifs are rendered incapable of binding Giα. BRET was observed with AGS3‐YFP and Giα1‐RLuc or with AGS3‐RLuc and Giα1‐YFP pairs. The latter donor and acceptor pair exhibited a significantly stronger net BRET signal potentially reflecting the assembly of a Giα1‐YFP acceptor on each of the four GPR docking sites in an individual AGS3‐RLuc molecule. These data indicate a robust interaction of GPR proteins and Giα1 in the living cell and provide a platform to determine how this interaction is regulated and where it occurs within the cell.
Interaction of AGS4 and Gialpha1 in living cells
Authors: Oner Sukru Sadik , Breton Billy , Bouvier Michel , Blumer Joe
Published: June 2021

Activator of G Protein Signaling‐4 (AGS4) is one of several receptor independent activators of G‐protein signaling identified in a yeast‐based functional screen of mammanlian cDNAs. AGS4 encodes a protein of 160 amino acids that contains 3 G‐protein regulatory (GPR) motifs each of which can serve as a docking site for Giα‐GDP free of Gβg. AGS4 and related GPR proteins provide unexpected regulatory mechanisms for G‐protein signaling systems. AGS4 is of particular interest in this regard as it is primarily expressed in the immune system. In addition to defining the functional role of AGS4 in immune cells, two of the key questions for AGS4 are what controls the interaction of these proteins with G‐protein and where does this interaction occur within the cell. As an initial approach to this question, we evaluated the interaction of AGS4 with Giα1 in living cells (HEK‐293) using bioluminescence resonance energy transfer (BRET) to measure interaction between proteins tagged with renilla luciferase (RLuc) and the YFP‐variant Venus. Cells transfected with AGS4‐YFP and Giα1‐RLuc exhibited a net BRET signal that was saturable and specific. The BRET signal was blocked by Gβγ expression and it was not observed with the AGS4‐Q/A mutant in which each of the GPR motifs are rendered incapable of binding Giα. In subsequent experiments, we examined the influence of various signaling systems on the AGS4‐Giα1 interaction. These data indicate a robust interaction of AGS4 and Giα1 in the living cell.
Regulatory effect of prostaglandin E2 on protein expression in bovine retinae, ex vivo
Authors: Opere Catherine A , Zhao Min , Robley Danielle , Braig Elizabeth , Kotera Casey , Ohia Sunny
Published: June 2021

We have evidence that prostaglandin (PG)E2 reduces endogenous bovine retinal amino acid neurotransmitter levels, ex vivo [Zhao et al, Methods and Findings, In press]. It is, however, unclear if these changes in neurotransmitter levels involve an effect on protein expression. Freshly isolated bovine eyeballs were injected, intravitreally with PGE2 and retinae were isolated and prepared for determination of protein expression using the 2DE‐DIGE approach. Protein spots exhibiting statistically significant changes (P<0.05) were selected and identified using LC‐MS/MS and analyzed by bioinformatics. PGE2 (10 uM) increased the expression of three proteins while five were decreased. The structural proteins, albumin and tubulin alpha‐1D chain and the cellular defense protein, heat shock 70 kDa protein 1B were up‐regulated. In contrast, peroxiredoxin 2 (a scavenger for peroxides) and aldehyde dehydrogenase 9 family, member A1 (an enzyme that breaks down alcohol) were down‐regulated. Furthermore, the GDP dissociation inhibitor 2, a signal transduction protein, recoverin, a calcium‐binding protein in photoreceptor cells and CNDP dipeptidase 2 were also down‐regulated. In conclusion, PGE2 altered the expression of both structural proteins and those involved in cellular defense and signal transduction pathways in bovine retina, ex vivo.
Arsenic trioxide inhibits p21 expression through the functional domains of TGIF
Authors: Huang Huei‐Sheng , Liu Zi‐Miao
Published: June 2021

Arsenic trioxide (ATO) has been used in medicine for many years, and is especially important in the treatment of acute promyelocytic leukemia. Multiple mechanisms of action, including cell cycle arrest and apoptosis, have been proposed; however, the opposing effects of arsenic in cancer therapy remain controversial. We found that ATO‐induced activation of p21WAF1/CIP1 (p21) led to A431 cell death through the antagonistic effects of the ERK1/2 and JNK1 signaling. Further, we noted that JNK induced phosphorylation of c‐Jun to recruit the TGIF/HDAC1 at the Sp1 sites, resulting in the suppression of p21 expression. Using the GST pull‐down assay and fluorescence resonance energy transfer analysis, we found the N‐terminal domain (amino acids 1‐108) of TGIF was critical to its binding with c‐Jun. In addition, using reporter assays, we observed that the C‐terminal domains (amino acids 138‐272) of TGIF, are essential to the suppression of ATO‐induced p21 expression. Thus, the domains of TGIF which carry out its inhibitory effects were identified.
Silencing of histone acetyltransferase GCN5 affects gene expression in human hepatoma cells: A gene array analysis
Authors: CHOUDHURY MAHUA , Pandey Ravi S , Clemens Dahn L , Davis J Wade , Lim Robert W , Shukla Shivendra D
Published: June 2021

A molecular understanding of the epigenetic control of gene transcription in cancer is essential for the development of therapeutic tools. Histone acetyltransferases (HATs) have been implicated in cancer and other diseases. The transcriptional regulator GCN5 is a HAT involved in acetylation of core histone, an important event in transcriptional regulation through alterations in the chromatin structure in eukaryotes. The role of GCN5 in liver is poorly known. We have therefore examined the transcriptional significance of GCN5 by knocking down its expression with siRNA in human hepatoma HepG2 cells derived VA‐13 cells. Illumina gene microarray analysis revealed that 956 genes are differentially expressed (≥1.5 fold up‐ or down‐regulated; statistically significant) due to GCN5 silencing. Among these 529 genes were up‐ and 427 were down‐regulated. GCN5 silencing up‐regulated antiapoptotic genes, e.g. ALS2CR2 (6.4 fold). On the other hand it down‐regulated pro‐apoptotic genes, e.g., CASPASE3 (3.5 fold), MMP7 (3.1 fold), and SLC2A3 (3.1fold). Gene involved in chromosomal stability, FAT10/UBD was also down‐regulated by 3.7 fold. In summary, these results provide evidence for a potential role of GCN5 in the expression of multiple genes in the human hepatoma cells.
Serotonin 5‐HT1A‐like, 5‐HT2, and 5‐HT7 Receptors Modulate Learning and Memory in Drosophila
Authors: Johnson Oralee , Becnel Jaime , Nichols Charles D
Published: June 2021

Serotonin is a neurotransmitter involved in many behaviors in mammals including regulation of mood, appetite, sleep, memory, aggression, and sexual behavior. In the fly, serotonin has also been demonstrated to be involved in many of these same complex behaviors. The effects of serotonin are primarily mediated through interactions with G‐protein coupled receptors to initiate multiple signal transduction pathways. Drosophila expresses orthologs of four mammalian 5‐HT receptor subtypes: 5‐HT1ADro, 5‐HT1BDro, 5‐HT2Dro, and 5‐HT7Dro. Here, we present data demonstrating involvement of all three serotonin receptor families in olfactory learning and memory in the fly.

We probed the involvement of the 5‐HT1A‐like, 5‐HT2, and 5‐HT7 receptors in short‐term memory in the fly. The 5‐HT1A receptor agonist U92106A and the 5‐HT1A receptor antagonist WAY100635 reduced performance indices in a dose‐dependent manner. The 5‐HT2 receptor agonist DOI, and the 5‐HT2 receptor antagonist ketanserin also abated performance indices in a dose‐dependent manner (IC50 value for DOI = 0.21 mM). A putative 5‐HT7 receptor antagonist SB 269970, also diminished performance levels. Since both agonists and antagonists for a given receptor inhibit performance, a homeostasis likely exists at each receptor, and perturbation of homeostasis disrupts this behavior.

Research support: NIMH grants 5R21MH078454‐02 and 3R21MH078454‐01A1S1
Effects of Memantine on NMDA Induced Long‐Term Depression in Mouse Models of Down Syndrome
Authors: Scott‐McKean Jonah Jacob , Smith Karen E , Dell'Acqua Mark L , Costa Alberto C. S.
Published: June 2021

The Ts65Dn mouse is the most complete animal model for Down syndrome. Recently, we have shown that the Ts65Dn mouse displays pharmacological responses consistent with a dysfunction in molecular pathways coupled to the gating of NMDA receptors; these include the finding that the uncompetitive NMDA receptor antagonist memantine rescues learning and memory deficits in these mice. In the present study, we used electrophysiological and biochemical techniques to probe potential mechanisms underlying these observations. NMDA mediated long‐term depression (LTD) was induced chemically (Chem‐LTD) in the CA1 region of acute hippocampal slices from Ts65Dn and euploid control mice. We found that Ts65Dn mice showed a greater synaptic depression when compared to euploid littermate controls. To determine whether memantine could rescue this phenotype in the Ts65Dn mice, half of the hippocampal slices were treated with memantine before Chem‐LTD treatment. We found that memantine‐treated hippocampal slices from Ts65Dn mice presented control levels of Chem‐LTD. Currently, phosphorylation of the AMPA receptor GluR1 subunit is being assessed on hippocampal slices from Ts65Dn and control mice, which were flash frozen under control and Chem‐LTD conditions, with or without drug. This study confirms our previous finding that molecular pathways coupled to the gating of NMDA receptors are altered in the Ts65Dn mouse.
SB‐269970, a selective 5‐HT7 antagonist, reverses working memory deficits in rats by normalizing glutamate neurotransmission
Authors: Galici Ruggero , Boggs Jamin D , Aluisio Leah , Fraser Ian C , Bonaventure Pascal , Lovenberg Timothy W
Published: June 2021

5‐HT7 antagonists have been shown to have efficacy in animal models of cognition. However, the mechanism by which these procognitive effects occur is not well understood. The goal of these studies was to evaluate the effects of SB‐269970, a selective 5‐HT7 antagonist, in translational models of cognition and establish whether it modulates glutamate neurotransmission. The effects of SB‐269970 (10 mg/kg, i.p.), were evaluated in the delayed non‐matching to position (DNMTP) task alone and in combination with MK‐801 (0.1 mg/kg, i.p.), a non‐competitive NMDA receptor antagonist, and, in separate experiments, with scopolamine (0.06 mg/kg, i.p.), a non‐selective muscarinic antagonist. Also, the ability of SB‐269970 (10 mg/kg, i.p.) to modulate MK‐801 (0.1 mg/kg, i.p.)‐induced glutamate and dopamine cortical release was evaluated using microdialysis in freely moving rats. MK‐801 and scopolamine significantly reduced percentage correct responding in the DNMTP task without affecting number of trials. SB‐269970 significantly reversed the deficits induced by MK‐801, but not by scopolamine. SB‐269970 normalized MK‐801‐induced glutamate but not dopamine release in the cortex and these effects were similar to LY379268, a group II mGluR agonist. These results indicate that blockade of 5‐HT7 receptors selectively normalizes glutamatergic neurotransmission.
Vulnerability to cocaine abuse in socially housed female monkeys: evidence for sex differences
Authors: Nader Michael A. , Riddick Natallia V. , Czoty Paul W. , Icenhower Michelle , Gage H. Donald
Published: June 2021

Earlier work in male monkeys reported that dopamine (DA) D2 receptor function changed as a consequence of social status and that low D2 receptor levels were associated with cocaine reinforcement. One goal of the present study was to extend these observations to female monkeys. Several variables were measured when monkeys were individually housed to determine if they predicted social rank. Monkeys that eventually became subordinate showed shorter latencies to touch a novel object and had higher basal CSF levels of a serotonin (5‐HT) metabolite (5‐HIAA). In brain imaging studies, DA D2 receptor and 5‐HT transporter availability did not predict eventual social rank. However, D2 receptor availability increased by ~ 20% when monkeys became dominant, an effect almost identical to that seen in male monkeys. When female monkeys self‐administered cocaine (0.001‐0.1 mg/kg per injection) under a fixed‐ratio schedule, dominant females acquired cocaine reinforcement at lower doses than subordinates. Furthermore, peak response rates were higher for dominant animals. These findings are opposite to those observed in socially housed male monkeys and suggest a direct relationship between social hierarchy‐induced changes in D2 receptor levels and cocaine reinforcement in females. The mechanisms accounting for these sex differences are currently under investigation. DA 17763.
An Elevated Fibrinogen Increases Matrix Metalloproteinases Activity in Cardiac Microvascular Endothelial Cells
Authors: Patibandla Phani Kumar , Tyagi Neetu , Tyagi Suresh C. , Dean William L. , Roberts Andrew M. , Lominadze David
Published: June 2021

Cardiovascular diseases such as hypertension, diabetes, and stroke are accompanied by increased blood fibrinogen (Fg) content. Increased levels of Fg is considered to be a high cardiovascular risk factor. We previously showed that elevated concentration Fg causes arteriolar constriction through release of endothelin‐1 (AJP, 2005; 288: H1257‐H1264) and increases endothelial cell (EC) layer permeability through formation of filamentous actin (Mol. Cell. Biochem, 2008; 307: 13‐22). To further investigate the mechanisms of Fg effects on microvascular permeability, we tested the hypothesis that Fg may be involved in vascular remodeling by activating matrix metaloproteinases (MMPs). Rat cardiac microvascular endothelial cell (RMVEC) cultures were treated with one of the following for 24 h: Fg (2 or 4 mg/ml), Fg (4 mg/ml) with MMP inhibitor GM6001 (1 μM), or medium (control). Activation of MMPs in the subendothelial matrix after addition of MMP substrate was assessed by measuring fluorescence intensity of the medium using a fluorometer. Activation of MMP9 was determined by an MMP9‐specific ELISA kit. Fg caused a dose‐dependent increase in activation of MMPs in the medium (97 ± 6 and 137 ± 12 fluorescence intensity units, FIU with 2 and 4 mg/ml Fg respectively, vs. 82 ± 6 FIU for control, mean±SE). Formation of active MMP9 was increased by 4 mg/ml of Fg (0.054 ± 0.001 ng/ml vs. 0.034 ± 0.004 ng/ml for control). Activation of MMPs by this dose of Fg was inhibited by GM6001, a nonspecific MMP inhibitor. These data suggest that an increased level of Fg induces activation of MMPs, and thus, may be a mechanism for the Fg‐induced increased EC layer permeability seen earlier.

Supported by NIH grant # HL080394 to DL.
VEGFR2 Receptor Cleavage, Endothelial Apoptosis and Capillary Rarefaction in the Spontaneously Hypertensive Rat
Authors: Tran Edward D , Schmid‐Schönbein Geert W
Published: June 2021

The spontaneously hypertensive rat (SHR) has a multitude of microvascular complications including endothelial apoptosis with capillary rarefaction. Our recent evidence shows that the SHR has elevated levels of proteolytic (matrix metalloproteinases, MMPs) activity and apoptosis in microvascular cells, but the molecular mechanism for the apoptosis is unknown. Our hypothesis is that selected MMPs cleave vascular endothelial growth factor receptor‐2 (VEGFR2), which in turn causes endothelial apoptosis and capillary rarefaction. Significantly enhanced levels of MMP‐1, ‐1/9, ‐2, ‐7, and ‐8 activities were observed in SHR plasma. The SHR also had a significantly lower expression of extracellular domains of VEGFR2 in cardiac microvessels. Furthermore, application of plasma from the SHR, or purified MMP‐1, ‐7, and ‐9 to natïve cells caused cleavage of the extracellular domain of VEGFR2. This receptor cleavage was blocked by broad acting MMP inhibitors (GM6001 1 µmol/L, EDTA 10 mmol/L, or doxycycline 11.3 µmol/L). Chronic MMP inhibition (doxycycline, 5.4 mg/kg/day, 24 weeks) attenuated VEGFR2 cleavage, endothelial apoptosis, and capillary rarefaction in the SHR. The results suggest elevated microvascular MMP‐1, ‐1/9, and ‐7 activities that may cause cleavage of VEGFR2, endothelial apoptosis, and capillary rarefaction in this model of hypertension. Supported by HL 10881.
heparin inhibits injury‐induced femoral artery remodeling in mouse Via CD44
Authors: Zhao Gaofeng , Kolliputi Narasaiah , Zhao Hang , Beagle John , Yu Lunyin , Syrkina Olga , Leu Shawwei , Hicham Mrabat , Hales Charles
Published: June 2021

Background

Previous studies have indicated heparin which has both anti‐coagulant and anti‐proliferative properties, may be used as a possible therapeutic agent for the treatment of post‐angioplasty restenosis. CD44 is the receptor of hyaluronan and plays essential roles in many aspects including SMC proliferation, inflammation and injury repair. This study investigates the effects of heparin on injury‐induced femoral artery remodeling and the expression of CD44 , to illuminate the molecular mechanism by which heparin blocks injury‐induced femoral artery stenosis.

Hypothesis

Heparin decreases injury‐induced femoral artery remodeling through the CD44 signal pathway,

Methods

The injury‐induced artery remodeling model was prepared in wild‐type and CD44 knock‐out mice by inserting a straight spring wire into the femoral artery via arterioctomy in a small muscular branch. Then mice were treated with heparin by subcutaneous injection at a dose of 20mg/kg once a day. 4 weeks later, we sacrificed the mice for assessment of histology of the injured femoral artery and for the expression of CD44.

Results

In wild‐type mice, heparin significantly decreased femoral artery remodeling, and western blot showed heparin could markedly promote CD44 expression in the injured arteries. In contrast with wild‐type mice, CD44 deficient mice demonstrated considerable greater arterial remodeling, which when treated with heparin, no longer showed any significant reduction in vascular remodeling.

Conclusion

Heparin can decrease injury‐induced femoral artery remodeling, at least partially, by upregulating the expression of CD44.
Decreased arterial distensibility in patients with systemic lupus erythematosus
Authors: Lanphere Kathryn Rosie , Sharrar Janeen M , Sibbitt Wilber L , Roldan Carlos R , Greene Ernest R
Published: June 2021

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease that is atherogenic. Arterial distensibility (AD) is a risk factor and an independent predictor of cardiovascular morbidity and mortality. Importantly, AD can be easily determined noninvasively with hand held, portable, low power, and relatively inexpensive equipment. Operator training is required, but minimal. Nevertheless, an association between SLE and AD has not been reported. Accordingly, we tested the hypothesis that patients with SLE would have a significantly (p<0.05) decreased AD when compared to healthy controls. We used noninvasive, high spatial resolution, 10 MHz, image‐guided, M‐mode sonography to measure maximal systolic and minimal diastolic diameters and diastolic intima‐media thickness (IMT) at 3 sites in the right common carotid artery of 17 SLE patients and 51 healthy age and gender matched controls. In the one blinded observer, the 95% confidence levels for accuracy and precision for noninvasive tonometric blood pressures and sonographic diameters were ~5mmHg and ~0.15mm, respectively. The calculated values for AD were determined by standard arterial mechanical models and algorithms. Results are given as X/SD with *= p<0.05 versus controls:

These preliminary data suggest that AD is significantly (p<0.05) decreased in young patients with SLE. Thus, arterial distensibility may be an efficient and inexpensive marker of clinically important atherosclerosis in young patients with systemic lupus eyrthematosus.

(Funded, in part, by NIHRO1HL077422)
Platelet activation is reversible and dependent on the shear stress‐exposure time integral
Authors: Rubenstein David A , Yin Wei
Published: June 2021

Our goal was to determine whether platelets have a memory for shear induced activation and if this is reversible. During cardiovascular diseases, such as atherosclerosis, platelets are subjected to complex pathological shear stress waveforms at particular vascular locations. Platelets may pass this location every circuit inducing a predisposition towards activation. We hypothesized that platelet activation was governed by the shear stress‐exposure time and not the absolute shear stress level. To test this, platelets were exposed to tailored shear stress waveforms to examine if platelets remember past shear stress histories. Activation was quantified with a prothrombinase assay, flow cytometry and aggregometry (agonized with TRAP6). Our prothrombinase assay depends on FVa and PS expression. To verify this assay and aggregation, CD62P, Annexin V and CD41/61 expression was measured. Platelet activation was waveform independent, if the shear stress‐exposure time was the same. This was true even for platelets subjected to supra‐critical shear stress levels (Hellum's curve). With increasing shear stress‐exposure time, activation was enhanced. Platelets that were subjected to a supra‐critical shear stress early were not more susceptible to subsequent shear exposures. Therefore, platelet activation is reversible and dependent on the shear stress‐exposure time and not the absolute value of shear stress.
Monocyte integrin CD11c/CD18 is a functional biomarker for risk of cardiovascular disease
Authors: Gower R Michael , Wu Huaizhu , Knowlton Anne A , Ballantyne Christie M , Simon Scott I
Published: June 2021

Atherosclerosis is a systemic disease in which hyperlipidemia, hemodynamics, and inflammation superpose to enhance monocyte recruitment to distinct arterial regions. This in turn leads to plaque formation, vascular occlusion, and myocardial infarction. Recently we reported that integrin CD11c/CD18 cooperated with VLA‐4 in monocyte recruitment in apoE‐/‐ mice fed a high fat diet. Here we investigate if CD11c correlates with risk factors of cardiovascular disease (CVD). 60 healthy human volunteers (50% male, age 19‐66) were asked to fast overnight for 10 hours. Blood was drawn and donors were fed a 45% fat meal. 3.5hrs postprandial, donors returned for a second blood draw. Blood glucose, total cholesterol, HDL, triglycerides, and apolipoprotein B content were measured fasting and after the meal. Triglycerides increased significantly, but other constituents were unchanged. Resting and MCP‐1 stimulated levels of CD11c on monocytes were significantly increased after the fatty meal. Donors over 35yrs expressed significantly more CD11c compared to the younger group, which correlated with higher BMI and total cholesterol and lower HDL. We conclude hyperlipidemia associated with a high fat meal and age can upregulate monocyte CD11c, suggesting this adhesion molecule may serve as a biomarker for those at risk of CVD. This work was supported by HL082689 to S.I.S. and T32 HL086350‐01A1 to R.M.G.
Adipocyte derived factor(s) is suppressed in the visceral fat in MC4R‐/‐ mice
Authors: Xiang Lusha , Fuller William , Hester Robert L
Published: June 2021

Adipocyte derived factors (ADFs) are suggested to contribute to endothelial dysfunction and cardiovascular diseases. We have shown that in ob/ob mice (ob), a model of obesity, both subcutaneous (SF) and visceral fats (VF) produce ADFs and induce vasodilation by activating KATP channels. There is evidence that MC4R‐/‐ mice, another model of obesity, do not develop hypertension or renal dysfunction despite the accumulation of VF. We hypothesize that there is be a decreased production of ADFs in MC4R‐/‐ mice. 12‐16 week old wild type (WT), ob, and MC4R‐/‐ were used. The spinotrapezius muscles in WT mice were prepared using intravital microscopy for observation of arcade arterioles. VF or SF in ob mice and MC4R‐/‐ was collected and were separately incubated in PSS. The vasodilatory responses to fat conditioned PSS were tested in WT mice before and after treatment with glibenclamide. The vasodilatory responses to SF or VF conditioned PSS from ob mice were not significantly different, similar with the vasodilation induced by SF conditioned PSS from MC4R‐/‐. However, VF from MC4R‐/‐ lead to a significantly attenuated vasodilation as compared with the SF‐induced dilation (31±6% vs. 69±7% n=4). Glibenclamide inhibited the vasodilations induced by each of the fat conditioned PSS. These results suggest that ADFs are suppressed in the VF in MC4R‐/‐ mice, which may be responsible for the protected outcomes despite of obesity.