bluuebunny/crossref_metadata_2025 huggingface dataset:
The de novo and remodeling pathways of dipalmitoylphosphatidylcholine synthesis in lung epithelial cells are physiologically linked.
Authors: Butler Phillip L , Mallampalli Rama K
Published: June 2021

Lung surfactant is essential for life and contains a critical molecule, dipalmitoylphosphatidylcholine (DSPtCho). Deficiency of surfactant DSPtdCho leads to atelectasis, mechanical abnormalities, and cardiovascular impairment. DSPtdCho production occurs by a de novo pathway and a remodeling pathway. A new enzyme involved in the remodeling pathway has recently been characterized, termed Acyl‐CoA:lysophosphatidylcholine acyltransferase (LPCAT). Preliminary results indicate that overexpression of LPCAT in lung epithelia decreases de novo PC synthesis while having no significant effect on PtdCho mass, indicating tight homeostatic control of DSPtdCho metabolism. Overexpression of LPCAT inhibited activity of the enzyme catalyzing the final step in the de novo synthesis pathway, cholinephosphotransferase (CPT). LPCAT overexpression reduced immunoreactive levels of two variants that exhibit CPT function, CEPT and CPT, without altering mRNA transcript levels for the enzymes. Thus, LPCAT is a critical enzyme that controls surfactant production by downregulating CPT activity either at the level of protein stability or by mRNA translational efficiency. These results further suggest that the remodeling and de novo pathways are physiologically linked.
Glucose uptake is increased and glyceroneogenesis is reduced in denervated retroperitoneal adipose tissue from fasted rats
Authors: Frasson Danubia , Chaves Valeria Ernestania , Santos Maria Emilia Soares Martins , Garófalo Maria Antonieta Rissato , Kettelhut Isis do Carmo , Migliorini Renato Hélios
Published: June 2021

It has been know that adipose tissue (WAT) has three sources of glycerol‐3‐phosphate (G3P)generation, but the control of these processes has been little studied. To obtain more information about the control of G3P generation in WAT, we investigate the effects of WAT denervation on the in vivo uptake of glucose by the tissue, and on the acitivities of P‐enol‐pyruvate carboxykinase (PEPCK), a key enzyme of glyceroneogenesis, and of glycerokinase (GyK). Unilateral denervation of retroperitoneal WAT was performed 7 days before the experiments, using contralateral WAT as control. Denervation caused a 68% decrease in WAT norepinephrine content. Rates of glucose uptake (ng.mg tissue−1. min−1) were lower in fasted (0.82±0.068)than in fed (2.60±0.28) rats and increased in denervated tissues: fasted 1.30±0.19 and fed 4.25±0.47. GyK activity (nmol. mg of protein−1. min−1) decreased in fasted rats (0.33±0.01 vs fed 1.13±0.13) and were further reduced by denervation (fasted 0.22±0.01, fed 0.73±0.08). The marked activation of PEPCK (nmol. mg of protein−1. min−1)in fasted rats (13.88±0.98 vs fed 2.32±0.19) was reduced by denervation (fasted 5.50±0.25, fed 1.51±0.16). The data show that sympathetic nervous system control an adequate supply of G3P for triacylglycerol synthesis in WAT in both groups, with reciprocally changes in G3P generation via glycolysis and via glyceroneogenesis. Financial support: CNPq and FAPESP.
Chronic AICAR‐induced AMP‐dependent kinase (AMPK) activation exerts an anti‐lipogenic effect in isolated rat adipocytes
Authors: Gaidhu Mandeep Pinky , Anthony Nicole Marie , So Mandy , Fediuc Sergiu , Ceddia Rolando Bacis
Published: June 2021

In the present study, we investigated the effects of chronic AICAR‐induced AMP‐dependent kinase (AMPK) activation on lipid metabolism in isolated rat adipocytes. Isolated rat epididymal fat cells were incubated with AICAR (0.5mM) for 15h in vitro, or extracted from rats injected in vivo with AICAR for 14 days. We found that adipocytes exposed to AICAR elicited an ~80% reduction in FA uptake, which was accompanied by a ~3.5‐fold increase in FA oxidation. This indicates that FAs entering the adipocyte were being diverted towards the oxidative pathway. In accordance with these observations, we also found a reduction in acetyl‐CoA carboxylase (ACC) expression, which is a critical regulator of lipid synthesis and malonyl‐CoA content in the cell. We also observed that chronic AICAR exposure increased the lipolytic response under basal (~8‐fold) and epinephrine‐stimulated (~2‐fold) conditions as demonstrated by the release of free FAs in the medium. Here, we provide novel evidence that chronic AICAR‐induced AMPK activation reduces FA uptake, diverts metabolism of FAs towards oxidation, and promotes a lipolytic response. Altogether, these data indicate that chronic AMPK activation exerts an anti‐lipogenic effect in adipocytes, and may be a potential avenue for therapies targeted towards obesity and its related metabolic disorders. This research was funded by NSERC, CIHR, and CDA operating grants.
Adenovirus mediated alteration of phosphatidylethanolamine N‐methyltransferase expression in vivo
Authors: Lingrell Susanne , Jacobs Rene L. , Vance Dennis E.
Published: June 2021

Hepatic phosphatidylcholine (PC) is required for normal secretion of VLDL. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP‐choline) pathway, and the flux through this pathway is regulated by the activity of CTP: phosphocholine cytidylyltransferase (CT). Liver cells are uniquein that they can also synthesize PC via the sequential methylation of phosphatidylethanolamine, catalyzed by phosphatidylethanolamine N‐methyltransferase (PEMT). We have previously reported that Pemt−/− mice have impaired VLDL secretion that leads to mild steatosis. To further investigate regulation of lipid metabolism by PEMT, we produced adenoviruses (Ad) that expressed either the PEMT cDNA (Ad.PEMT) or a PEMT siRNA (Ad.siPEMT). In the first set of experiments, we observed that in vivo administration of Ad.PEMT to Pemt−/− mice normalized hepatic PEMT activity within 7 days. The restoration of PEMT activity in Pemt−/− mice returned hepatic TG to wildtype levels. Adenoviral overexpression of CTα also normalized hepatic TG in Pemt−/− mice. Although both Ad.PEMT and Ad.CT were successful in reversing steatosis in Pemt−/− mice, only CT overexpression increased plasma TG levels. In the second study, Ad.siPEMT was administered, by i.v. injection, to wildtype mice. After 7 days, the levels of PEMT mRNA, protein, and activity were reduced by 20 to 40% as compared to control mice. Although the suppression of PEMT was minor, a lowering of plasma TG was observed, suggesting that an acute decrease in PEMT was sufficient to alter lipid metabolism. The creation of these adenoviruses provides us with a valuable tool to investigate the influence of acute changes in hepatic PC biosynthesis on lipid and lipoprotein metabolism. Supported by Canadian Institutes of Health Research.
The complement protein properdin recognizes glycosaminoglycan chains on apoptotic and malignant cell surfaces
Authors: Hourcade Dennis , Mitchell Lynne , Zhang Lijuan , Kemper Claudia
Published: June 2021

The complement alternative pathway (AP) C3 convertase, a central player of the complement cascade, tags potential targets with C3 fragments that in turn direct lysis and/or clearance and inflammatory reactions. The complement protein properdin has long been known to stabilize the AP convertase. Recently we have found that properdin is also a danger‐recognition molecule: Properdin can bind malignant and apoptotic cells and certain pathogenic microbes. Once bound to a surface, properdin provides a platform for the assembly of the AP C3 convertase, leading to opsonization and clearance and/or lysis. We are interested the mechanism by which properdin can recognize such a variety of potential targets.

Properdin is composed of 6 thrombospondin repeats, which in other proteins can harbor glycosaminoglycan (GAG) binding sites. Thus, we investigated the possibility that properdin attaches to mammalian cells via GAGs. Using a panel of wild type and mutant CHO lines, we observed that properdin can bind cells via heparin sulfate and chondroitin sulfate GAG chains associated with cell surface proteoglycans. We then examined properdin interactions with human cells and found that properdin binds GAGs on apoptotic and malignant cell surfaces. Experiments with soluble GAG competitors suggest that properdin harbors multiple GAG‐binding sites that differ in GAG specificity. Supported by NIHR01AI05143.
Protein Scaffolds for Multivalent Display of High‐Affinity CD22 Ligands
Authors: O’Reilly Mary K. , Liao Liang , Rillahan Cory , Collins Brian E. , Han Shoufa , Kitov Pavel , Bundle David R. , Paulson James C.
Published: June 2021

CD22 is a sialic acid‐binding immunoglobulin‐like lectin (Siglec) that is expressed on the surface of B cells and functions as a regulator of B cell receptor signaling. The relationship between glycan binding and immunomodulatory function is being explored, and has led to the development of ligand‐based probes of CD22. CD22 binds to glycan ligands bearing the terminal sequence NeuAcα2‐6Gal with low affinity (Kd = ~0.2 mM), requiring multivalency to achieve stable binding. We have used a decavalent anti‐nitrophenol (NP) IgM antibody to display hetero‐bi‐functional ligands comprising a CD22 ligand coupled to the antigen NP. In this approach the protein scaffold participates in ligand‐driven assembly of multivalent IgM‐CD22 complexes, which can be demonstrated on immobilized recombinant CD22 by ELISA or flow cytometry of CD22‐immobilized magnetic beads. More importantly, assembly is observed on the surface of native B cells, as detected by immunofluorescence and flow cytometry, even though the ligand‐binding site of CD22 is largely masked by endogenous glycan ligands on the cell surface. This assembly is sensitive to the length and structure of the spacer, and also to the valency of the scaffold, as anti‐NP IgA and IgG display decreased avidity compared to anti‐NP IgM. The observation that a divalent protein scaffold succeeds where synthetic probes of higher valency do not validates this approach.
Modulation of GPCR Function by α‐Tocopherol
Authors: Bennett Michael P , Bergman Christian , Mitchell Drake C.
Published: June 2021

The function of vitamin E (vitE) is typically ascribed to protection from oxidation, however recent reports describe its non‐antioxidant functions. To investigate the non‐antioxidant effects of vitE in GPCR signaling, we reconstituted bovine rhodopsin into 1‐stearoyl‐2‐docosohexanoyl‐phosphatidyl choline (SDPC) bilayers. The effects of 5 and 10mol% vitE on rhodopsin function were determined spectroscopically by measuring the equilibrium amount of the signaling‐competent conformation of rhodopsin (MetaII) following photoactivation. The kinetics of MetaII formation was measured by flash photolysis. Effects of vitE on rhodopsin thermal stability were determined by differential scanning calorimetry (DSC). Phospholipid acyl chain packing was determined via DPH time‐resolved fluorescence polarization anisotropy. Addition of vitE reduced both the rate of MetaII formation and the amount of MetaII formed. DSC thermograms demonstrate that vitE increases the thermostability of rhodopsin by up to 0.8°C. Model‐dependent analysis of the scan rate‐induced shift in Tm shows that vitE slows the kinetics of rhodopsin denaturation. VitE induced more constrained acyl chain packing while reducing DPH fluorescence lifetimes. These results demonstrate that high levels of vitamin E modulate lipid packing and inhibit GPCR activation in SDPC bilayers.
Elimination of a homologous domain of an intestinal hydrolase enhances trafficking and affects polarized sorting via altered membrane association
Authors: Behrendt Marc , Naim Hassan
Published: June 2021

The transport‐competent precursor of human intestinal lactase‐phlorizin hydrolase, pro‐LPH, encompasses four homologous domains, whereby the domains III and IV represent the mature brush border enzyme (LPH[beta]). The influence of each of the homologous domains on the structural and functional characteristics of the pro‐LPH polypeptide has been analysed by directed restructuring of the domain composition. Removal of domain IV, which carries the lactase activity, results in accelerated transport kinetics and altered patterns of quaternary structure and membrane association. These novel biosynthetic features of the mutant ([Delta]IV) are directly associated with a retarded dimerization of this mutant in the Golgi and a reduced association with Tween 20‐resistant membranes in the ER. These non‐classical types of membrane microdomains have been shown to be implicated in an early sorting mechanism of membrane proteins. Another mutant that lacks domain III is partially folded and blocked in the ER. Domain III per se is transport competent, enzymatically active and efficiently sorted to the apical surface. Altogether, our data strongly suggest a regulatory role of the membrane associated domain IV in the trafficking of pro‐LPH, while domain III comprises all necessary information for apical sorting.

This work has been supported by the German Research Council.
Alpha‐1A adrenergic receptors regulate neurogenesis and cognitive function
Authors: Boese Sarah Jane , Schlosser Danielle D , Knudson Chris A , Carr Patrick A , Perez Dianne M , Doze Van A
Published: June 2021

A major target of the adrenergic system is the hippocampus, a region that is critical for learning and memory. Recent studies suggest alpha‐1A adrenergic receptors (α1A‐ARs) may regulate neurogenesis and neuronal differentiation. Our understanding of the function of α1A‐ARs is limited due to a lack of specific ligands and antibodies. To address this, transgenic mice were generated which over‐express the α1A‐AR with enhanced green fluorescent protein (EGFP) or constitutively active mutant (CAM) α1A‐AR. Knockout (KO) α1A mice were also generated. Immunohistochemistry showed that CAM α1A‐AR mice had increased BrdU incorporation compared to normal and KO α1A‐AR mice. Increased numbers of hippocampal interneurons in CAM α1A mice compared to normal mice were also observed. Increased interneurons may affect learning and memory. Normal, CAM α1A, and KO α1A mice were tested on a multi‐component T‐maze and the Morris water maze. CAM α1A mice displayed increased cognitive ability in the T‐maze and the Morris water maze compared to normal mice. In both models, KO α1A‐AR mice displayed the worst cognitive ability. Treating normal mice with the selective α1A‐AR agonist cirazoline also showed enhanced learning and memory processes. Stimulation of α1A‐ARs may offer a new therapeutic strategy for increasing cognitive function and treating neurodegenerative diseases. Supported by NIH, NIH COBRE, NSF ND EPSCoR, NSF CAREER.
Few residues within extensive binding interface determine receptor preference of arrestin proteins
Authors: Vishnivetskiy Sergey A. , Francis Derek , Klug Candice S. , Gurevich Vsevolod V.
Published: June 2021

The “footprint” of light‐activated phosphorhodopsin (P‐Rh*) on rod arrestin covers much the concave surfaces of the two domains. We used site‐directed spin labeling EPR spectroscopy to map the receptor‐binding surface of non‐visual arrestin2. The mobility of 15 residues (out of 21 tested) was reduced upon receptor binding, indicating that these positions are at or near the interaction interface. The receptor‐binding site in arrestin2 is as extensive as the rhodopsin‐binding site of rod arrestin. Taking advantage of a 4‐5‐fold difference between visual arrestin and arrestin2 binding to P‐Rh*, we used a gain‐of‐function approach to mimic rod‐like arrestin on the background of arrestin2. The combination of 5 and 8 mutations of exposed residues in the N‐ and C‐domains, respectively, corresponding to those present in rod arrestin reverses receptor preference of arrestin2. The resulting mutant binds P‐Rh* as well as wild type rod arrestin. Subsequent analysis by “loss‐of‐acquired‐function” approach, in which we mutated individual residues back to those present in arrestin2, identified one position in the N‐domain and three in the C‐domain that are primarily responsible for receptor discrimination. Thus, out of >40 amino acids in the ∼20x60A interaction interface, four exposed residues largely determine receptor specificity of arrestin proteins. NIH grants EY11500, GM 77561 (VVG), AI58024, GM70642 (CSK).
Electrophysiological characterization of derivatized alpha‐hemolysin for single‐molecule fluorescence studies in planar lipid membranes
Authors: Taylor Madison Michael , Bailey Derek J. , Paulson Ashley R. , Keranen‐Burden Lisa M. , Burden Daniel L.
Published: June 2021

Previous biochemical and crystallographic investigations have established that Staphylococcus aureus alpha‐hemolysin (αHL) forms conductive pores in lipid membranes by self‐assembling in a series of steps from seven identical protein monomers. However, the assembly process, the translational dynamics of the pre‐pore and pore, and the quaternary structure of αHL in lipid bilayers have not been directly visualized. Furthermore, visualization at the single‐molecule level has not been simultaneously correlated with transmembrane current. Here, we report findings from electrical studies on a battery of eight site‐directed mutants that are covalently linked to one of two fluorescent dyes (BODIPY‐TMR or TMRIA). Ensemble and single‐channel current measurements are performed using horizontal diphytanoyl phosphatidylcholine lipid bilayers. Channel conductance, voltage rectification ratio, and gating stability are used to compare mutants, fluorescently labeled mutants, and wild‐type αHL. All mutated and labeled channels maintain pore‐forming capability, although differences in conductance state, rectification ratio, and gating stability are observed. We present details of the electrical characterization and discuss initial work on simultaneous optical and electrical measurements in planar lipid membranes.
Structural Studies of Steap3, the Human Erythroid Ferric Reductase, Suggest a Gating Mechanism for Electron Transfer Across the Endosomal Membrane
Authors: Lawrence C. Martin , Sendamarai Anoop K. , Ohgami Robert S. , Fleming Mark D
Published: June 2021

The daily production of 200 billion erythrocytes requires 20 mg of iron, accounting for nearly 80% of the iron demand in humans. Thus, erythroid precursor cells possess an efficient mechanism for iron uptake in which iron loaded transferrin (Tf) binds to the transferrin receptor (TfR) at the cell surface. The Tf:TfR complex then enters the endosome via receptor mediated endocytosis. Following acidification within the endosome, iron is released from Tf and reduced from Fe3+ to Fe2+ by Steap3, the endosomal erythrocyte ferrireductase. The reduced iron then undergoes transport across the endosomal membrane by divalent metal iron transporter 1 (DMT1). Steap3 is a member of a unique family of metalloreductases. These proteins are comprised of a C‐terminal transmembrane domain thought to coordinate a single intramembrane heme and, with the exception of Steap1, an N‐terminal cytosolic oxidoreductase domain. Cytosolic NADPH and a flavin are used as cofactors, but the NADPH/flavin binding domain differs significantly from those in the bacterial Fre, yeast FRE, plant FRO or GP91phox families of metalloreductases. Instead, Steap3 shows limited similarity to the F420H2:NADP+ Oxidoreductase from A. fulgidus. We have determined the crystal structure of the Steap3 oxidoreductase domain in the absence and presence of NADPH. These structures suggest a gating mechanism for electron transfer across the endosomal membrane.
Mutagenesis of potential transmembrane palmitoylation sites reduces the surface expression of a chimeric membrane receptor
Authors: Velez‐Carrasco Wanda , Soto Coral , Eterovic Vesna , Alexander John , Green William N , Hann Richard M
Published: June 2021

A chimeric receptor with an alpha7 nicotinic receptor extracellular segment & serotonin type 3A receptor transmembrane (TM) & cytosolic (CY) segments, when transiently transfected into human tsA201 cells, is highly palmitoylated, which appears to promote cell‐surface & functional expression (Drisdel et al. 2004). The TM & CY segments contain 5 potential palmitoylation sites: C231 & C278 in TM domains M1 and M3, respectively, & C322, C344 & C357 in the M3–M4 CY loop. Cys‐to‐ala mutants were transiently expressed in tsA201 cells & the number of surface & intracellular receptors & surface receptor function were assessed. Surface expression of receptors with the double TM segment mutation C231A/C278A was only 48% that of wild‐type (WT) receptors (p=0.01), while expression of receptors with the triple CY loop mutation C322A/C344A/C357A did not differ significantly from WT. Expression of C278A was 81% of WT (p=0.02) while expression of C231A did not differ significantly from WT. Mutants C278A/C322A/C344A/C357A & C231A/C278A/C322A/C344A/C357A both showed significantly lower surface expression than WT (47%, p=0.005 & 29%, p=0.05, respectively). The amount of intracellular receptors produced in cells transfected with the above TM segment mutants did not differ significantly from that of WT, suggesting that removal of sulfhydryl groups in the TM segments interfered with the trafficking of fully‐assembled receptors to the cell surface. Cells expressing the 5‐cysteine mutant receptor accumulated 2‐fold higher intracellular calcium levels than cells expressing WT (p<0.001). [NINDS/SNRPMI 5U54NS39408 (VAE); T32 DA07255 (JA); NS043782, DA13602, NS043782, & DA019695 (WNG); NIGMS/MBRS 2SO6GM50695 (RH)]
An Escherichia coli LptA Mutant Defective in Lipopolysaccharide Export
Authors: Ma Bing , Raetz Christian R.H.
Published: June 2021

Lipopolysaccharide precursors and phospholipids in Escherichia coli are synthesized on the inner surface of the inner membrane and are transported through the periplasm to the outer surface of the outer membrane by a mechanism that is poorly characterized. Some evidence suggests that a periplasmic protein LptA, a cytosolic protein LptB and a membrane‐bound protein LptC are involved in lipopolysaccharide export, but their roles are not clearly defined. We now report a temperature‐sensitive mutant, MB1, with Ser62 → Cys and Gln111 → Pro substitutions in LptA. As shown by 32P‐ and 35S‐labeling, export of phospholipids and newly synthesized proteins in MB1 were not impaired, but the lipopolysaccharide export to the outer membrane is largely inhibited after 30 min at 42 ° C. Using the lipid A 1‐phosphatase LpxE as the periplasmic marker and the lipid A 3‐O‐deacylase PagL as the outer membrane marker, we were able to determine that the lipid A in MB1 at 42 ° C was terminally localized to the periplasm because it was dephosphorylated by LpxE but was not deacylated by PagL. Moreover, transmission electron microscopy showed that dense particles accumulated at the periplasmic space in MB1 at 42 ° C, consistent with a key role for LptA in lipopolysaccharide trafficking at a step subsequent to the inner membrane flip‐flop but prior to the outer membrane flip‐flop. This research was supported by National Institutes of Health Grant GM‐51310 to C. R. H. Raetz.
Brefeldin A‐inhibited guanine nucleotide‐exchange protein 1 (BIG1) associates with nucleolin and U3 snoRNA in HepG2 cell nuclei
Authors: Padilla Philip Ian , Uhart Marina , Peculis Brenda , Moss Joel , Vaughan Martha
Published: June 2021

BIG1, a brefeldin A‐inhibited guanine nucleotide‐exchange protein, activates class I ADP‐ribosylation factors (ARF1–3) by catalyzing replacement of bound GDP by GTP, an action critical in secretory transport from endoplasmic reticulum to cis‐Golgi. Our earlier report that BIG1 concentrated in nucleoli of serum‐starved HepG2 cells (Padilla et al., 2004) prompted us to identify molecules associated with BIG1 in the nucleolus. Antibodies against BIG1 co‐precipitated nucleolin (No, an abundant nucleolar protein) from nuclei; likewise, antibodies against No would co‐precipitate BIG1. Incubation of nuclei with RNAse or DNAse abolished co‐immunoprecipitation of BIG1 and No indicating the interaction depended on nucleic acids. Analysis of small nucleolar (sno)RNAs immunoprecipitated with BIG1 and No from nuclei revealed predominant bands of ~210 and ~70 bases; hybridization of the larger with U3‐specific oligonucleotides confirmed its identity. Cloning of U3 snoRNA cDNAs from the material precipitated by antibodies against BIG1 or No yielded identical nucleotide sequences. Continuing characterization of the mechanism and function of BIG1 accumulation in nucleoli of serum‐deprived cells may facilitate their comparison with those of the rapid nuclear accumulation of BIG1 that resulted from protein kinase A‐catalyzed phosphorylation of BIG1 Ser 883. Supported by Intramural Research Program, NHLBI, NIH.
The Integration of the Wnt and cAMP Signalling Pathways by AKAP220
Authors: Logue Jeremy Scott , Scott John D
Published: June 2021

The cAMP‐responsive protein kinase PKA is a multifunctional enzyme with a broad range of substrates. Proteins designated A‐kinase anchoring proteins (AKAPs) coanchor PKA and substrates together to enhance PKA signalling specificity. Members of this family include MAP2 (microtubule‐associated protein 2), neuronally expressed AKAP79 and AKAP150, and the DNA binding AKAP95. AKAP220 is a less‐well characterized A‐kinase anchoring protein however, is known to anchor protein phosphatase 1 (PP1), PKA, and glycogen synthase kinase‐3 (GSK‐3). Furthermore, I have recently identified β‐catenin as an additional anchored protein. β‐catenin is a transcription factor central to the Wnt signalling pathway. In the absence of Wnt signalling, GSK‐3 can phosphorylate β‐catenin to direct its rapid proteasomal degradation. It has been shown previously by Tanji et al. (2002) JBC, that GSK‐3 anchored to AKAP220 is phosphorylated by PKA and that phosphorylation inactivates GSK‐3. I have shown that the downstream effector of GSK‐3, β‐catenin forms a complex with over expressed AKAP220 in HEK 293 cells. Additionally, endogenous AKAP220 and β‐catenin form a complex in rat H9C2 and PC12 cells. Cellular based assays also suggest that PKA can phosphorylate β‐catenin following stimulation with the adenylyl cyclase activator forskolin. Our recent evidence therefore suggests that AKAP220 may integrate the cAMP and Wnt signalling pathways. The integration of cAMP signalling and the Wnt pathway may play a critical role in processes like wound healing and in pathological conditions like cardiac hypertrophy, of which both pathways have been implicated. The work described will provide fundamental information regarding the integration of these important pathways.

*This work is supported by National Institutes of Health grant DK54441.
Small angle x‐ray scattering and analytical ultracentrifugation characterization of the extracellular domain of α‐neurexin, alone and in complex with neuroligin‐1
Authors: Comoletti Davide , Miller Meghan T , Wilson Jennifer , Trewhella Jill , Taylor Palmer
Published: June 2021

Presynaptic cell adhesion protein α‐neurexin associates with neuroligins, its postsynaptic partner. Polymorphisms of the coding regions of neurexin and neuroligin genes were recently found to be associated with autism spectrum disorders and mental retardation. By small angle x‐ray scattering (SAXS) and analytical ultracentrifugation we have determined the basic structural parameters (maximum dimension, radius of gyration, molecular mass and volume) of the extracellular domain of α‐neurexin. Using the same data, we are now working on the determination their low resolution structure. Gel filtration chromatography and SAXS data suggest that the extracellular domain of α‐neurexin is monomeric in solution. Preliminary binding data on the α‐neurexin/neuroligin‐1 complex suggest a 2:2 stoichiometry where two α‐neurexin molecules bind to a neuroligin‐1 dimer. As mutations of neurexin and neuroligin genes appear linked to autism, these new structural models provide a framework for understanding altered recognition by these proteins in neuro‐developmental disorders.

This work was supported by: USPHS Grant R37 GM‐18360 and NIEHS to PT, NAAR #843 and CAN Pilot Research Award to DC; DOE/SC Grant and ARC Federation Fellowship to JT
Up Regulation of Estrogen Receptor alpha in ob/ob and Agouti Mice
Authors: Chakraborty Sanjoy , Boyer Amy , Yakov Tomer , Jadrayeva Saltanat
Published: June 2021

Increase in obesity is associated with reproductive failure. Estrogen levels affect reproduction and regulates appetite and food intake. Estrogen acts via its two receptors ERα and ERβ. ERα is necessary to maintain normal food intake, body weight and adiposity. Leptin plays a central role in regulating feeding behavior. The present study was undertaken to assess the hypothalamic distribution of ERα and ERβ in leptin‐deficient, obese, infertile ob/ob mice and infertile, obese, Ay/a agouti mice with high circulating leptin levels, in comparison to control mice. ERα‐immunoreactive cells in the arcuate nucleus and ERβ in the paraventricular nucleus were quantified. Stereologic analysis showed that there were significantly higher numbers of ERα‐immunoreactive cells in ob/ob mice irrespective of sex. Ovariectomy in female wild type mice caused a 50% increase ERα‐immunoreactive cells. The number of ERβ cells was the same among all the groups in both ARH and PVN. The difference in the number of ERα‐immunoreactive cells in Ay/a and ob/ob mice most likely does not reflect a decrease in circulating gonadal steroid hormones but most probably the acyclicity in there release. Our results suggest that although leptin and estrogen act via neuronal circuits to affect reproduction, neuroendocrine and behavioral processes, that circulating leptin may not directly regulate ERα levels in the hypothalamic arcuate neurons.
Temporal and tissue‐specific control of EC‐SOD expression: a new mouse model for examining the physiological and pathological roles of extracellular superoxide
Authors: Zou Yani , Chen Chih‐hsin , Huang Ting‐Ting
Published: June 2021

The extracellular isoform of superoxide dismutase (EC‐SOD, Sod3) plays a protective role against various injuries mediated by oxidative stress, including acute ischemia, stroke, and hyperoxia. In order to investigate the mechanisms underlining the physiological and pathological roles of EC‐SOD, we designed a Tet‐inducible EC‐SOD construct (TRE‐Sod3‐GFP) which contains a modified TRE promoter (TRE‐Tight), HA‐tagged Sod3 (EC‐SOD/HA), internal ribosome entry site (IRES), and a modified GFP (hrGFP). In vitro expression study showed non‐detectible baseline expression of EC‐SOD, but a robust expression in response to doxycycline induction. Transgenic mice generated from this construct were crossed to transactivator transgenic lines (CamKII‐tTA and LAP‐tTA) to test the expression of the transgene in the forebrain and liver. The results show that TRE‐Sod3‐GFP/CamKII‐tTA double transgenic mice express higher levels of EC‐SOD in hippocampus and cortex, whereas TRE‐Sod3‐GFP/LAP‐tTA have increased EC‐SOD in liver and plasma (as the secreted form). EC‐SOD protein level also correlates well with increased SOD activity in the plasma of TRE‐Sod3‐GFP/LAP‐tTA mice, suggesting that EC‐SOD produced from the transgene are biologically active. The Tet‐inducible TRE‐Sod3‐GFP mouse model will be helpful in interpreting the multiple biological roles of EC‐SOD in different organs. Funding from NIH and PAIRE.
Regulation of cell adhesion and motility: a novel function of arrestin proteins
Authors: Cleghorn Whitney Marie , Chen Dong , Zhang Xi , Zent Roy , Gurevich Vsevolod V.
Published: June 2021

Arrestins regulate G protein‐coupled receptors and other signaling proteins. Over 100 arrestin interaction partners have been identified, implicating arrestins in various signaling pathways and cellular functions. The interactions of many proteins (e.g., Src, JNK3, ERK½, Mdm2,etc.) with receptor‐bound arrestin localize these molecules to receptor‐rich membranes. Our recent finding that arrestins bind microtubules and recruit signaling proteins to the cytoskeleton prompted us to investigate whether arrestins affect cell adhesion, motility, and morphology. Here we show that the overexpression of arrestin2 and arrestin3 differentially regulates cell adhesion. Arrestin3 causes a significant reduction in cell adhesion to extracellular matrix protein fibronectin, whereas arrestin2 does not affect it. We also found that overexpression of arrestin2 and arrestin3 dramatically decreases migration, with arrestin2 showing the greatest reduction. Rhodamin‐phalloidin staining revealed that arrestin2 and arrestin3 induce strikingly different changes in cell morphology. Thus, the two non‐visual arrestins differentially regulate cell adhesion, spreading and migration. This is a novel biological role for arrestin proteins. NIH Grants GM77561 (VVG), DK69921 and VA Merit Award (RZ)
Angiotensin II modulates vascular smooth muscle cells alpha‐1D‐adrenoceptors
Authors: Gallardo‐Ortíz Itzell , Barajas Maximiliano Ibarra , López‐Sánchez Pedro , Villalobos‐Molina Rafael
Published: June 2021

Angiotensin II plays an important role in regulating systemic arterial pressure, provokes cardiovascular remodelling and hypertension, as well as an increase in alpha1‐adrenoceptors mRNA and protein. The aim of this work was evaluate the influence of angiotensin II on alpha 1D‐adrenoceptors in isolated smooth muscle cells of Wistar rat aorta. Smooth muscle cells were incubated with angiotensin II (100 nM) for different periods of time (30min–24hrs), and then protein by Westernblot was determined for the alpha 1‐adrenoceptor subtype.

The results show an important increment of alpha 1D‐adrenoceptors at 30 min by angiotensin II action, then a time‐dependent diminution in expression of protein at later measurement points. The maximal expression was blocked by losartan and ciclohexymide. This data suggesting that stimulation of AT1 receptors by angiotensin II in isolated cells conduce to increase of alpha 1D‐adrenoceptors and this effect is due to synthesis the novo of alpha 1D‐adrenoceptors, however there is not correlation with longer exposure of animals to the peptide, i.e., hypertension and hypertrophy, and suggest compensatory mechanisms in the animals not observed in isolated cells.

Supported by Conacyt 47481, PAPIIT IN203205 and Fundación Miguel Alemán
Caveolin‐1 Scaffolding Domain Regulates Store‐Operated Calcium Influx by Interacting with IP3R and TRPC1 in endothelial cells
Authors: Sundivakkam Premanand , Tiruppathi Chinnaswamy , Thippegowda Prabhakar , Kwiatek Angela , Sharma Tiffany T , Minshall Richard D , Malik Asrar B
Published: June 2021

Caveolin‐1 (Cav1) expression is required for store‐operated Ca2+entry in endothelial cells. We have shown previously Cav1 scaffolding domain (CSD) peptide interacted with the C‐terminus of TRPC1 and inhibited Ca2+ influx in endothelial cells (Mol Pharmacol.70:1174–1183, 2006). To understand the basis of Cav1 regulation of Ca2+ influx, we ectopically expressed CSD truncated Cav1 (Cav1ΔCSD) and TRPC1 C‐terminus truncated (TRPC1‐CΔ781–789) mutants in endothelial cells. We observed >2‐fold increase in thrombin‐induced store Ca2+ release‐activated Ca2+ influx in Cav‐1ΔCSD expressing cells compared to controls. In contrast, TRPC1‐CΔ781–789 mutant expression abolished thrombin‐induced Ca2+ influx. Immunoprecipitation studies revealed that WT‐Cav‐1 and WT‐TRPC1 interacted with endogenous IP3R. Whereas expression of TRPC1‐CΔ781–789 with WT‐Cav1 resulted in ∼50% reduction in the ability of IP3R binding to TRPC1. Interestingly, expression of Cav1ΔCSD with WT‐TRPC1 caused >80% reduction in the ability of IP3R binding to TRPC1. We also assessed the direct binding of IP3R with Cav1. Importantly WT‐Cav1 but not the Cav1ΔCSD interacted with endogenous IP3R. Co‐expression of TRPC1‐CΔ781–789 and WT‐Cav1 increased Cav1 binding to IP3R. These results suggest that Cav1 interacts with IP3R and TRPC1 through CSD to form IP3R‐Cav1‐TRPC1 complex and thus regulates store‐operated Ca2+ entry in endothelial cells.
EPAC promotes pathophysiological neointima formation in the rodent artery
Authors: Minamisawa Susumu , Yokoyama Utako , Hong Quan , Akaike Toru , Sata Masataka , Ishikawa Yoshihiro
Published: June 2021

Neointima formation (NF) is a characteristic feature of pathological (i.e. mechano‐injury) and physiological (i.e. vascular development) vascular remodeling. We have demonstrated that chronic stimulation of the prostaglandin E2 (PGE2)‐cAMP‐PKA signal pathway plays a critical role in physiological NF in perinatal ductus arteriosus (DA), a fetal bypass artery between the aorta and pulmonary artery. EPAC, a newly identified effector of cAMP, may play a distinct role from PKA in pathophsiology of NF. Quantitative RT‐PCR and immunoistochemical analyses revealed that the levels of EPAC1 were significantly upregulated in the rat perinatal DA and the mechanically injured artery. 8‐pCPT‐2′‐O‐Me‐cAMP, a cAMP analog selective to EPAC activator promoted smooth muscle cell (SMC) migration in a dose‐dependent manner. Adenovirus‐mediated EPAC1 gene transfer further enhanced 8‐pCPT‐2′‐O‐Me‐cAMP‐induced cell migration. In addition, transfection of siRNAs for EPAC1 significantly inhibited serum‐mediated SMC migration. Actin stress fibers were well‐organized with enhanced focal adhesion and cell shape was widely expanded in the presence of 8‐pCPT‐2′‐O‐Me‐cAMP. Adenovirus‐mediated EPAC1 gene transfer induced prominent NF in the rat DA explants. Thus EPAC promotes SMC migration and thereby pathophysiological NF.
Purification, sequencing, and molecular identification of a mammalian PP‐InsP5 kinase that is activated when cells are exposed to hyperosmotic stress
Authors: choi jae h , Williams jason , cho jaiesoon , falck j , shears stephen B
Published: June 2021

The “inositol pyrophosphates” have been reported to regulate vesicle trafficking, transcription, chemotaxis, telomere maintenance, apoptosis, DNA repair, and environmental stress responses. The family of kinases that synthesize PP‐InsP4 (diphosphoinositol tetrakisphosphate) and PP‐InsP5 (diphosphoinositol pentakisphosphate) have been cloned. A kinase activity that phosphorylates PP‐InsP5 to [PP]2‐InsP4 (bis‐diphosphoinositol tetrakisphosphate) has been observed in mammalian cell extracts, but this kinase(PPIP5K1; E.C. ) has not previously been cloned. In this study we describe the purification, sequencing and cloning of a mammalian PPIP5K. We demonstrate here that mammalian PPIP5K is a homologue of yeast Vip1 that functions primarily as a PP‐InsP5 kinase both in vitro and in intact cells. Moreover, we show that this enzyme is acutely activated by osmotic stress. This research was supported by the Intramural Research Program of the NIH / National Institute of Environmental Health Sciences.
Experimental and theoretical studies to determine smooth muscle nitric oxide availability
Authors: Namin Shabnam Mansourie , Tsoukias Nikolaos
Published: June 2021

Nitric Oxide (NO) is produced in the vascular endothelial cells (EC) in response to hemodynamic or agonist stimuli and diffuses to the smooth muscle (SM) to regulate vascular tone. In the microcirculation, the close proximity of the site of NO production to the red blood cells (RBC) and the fast consumption of NO by hemoglobin (Hb) suggests that most of the NO produced will be scavenged by the blood. At this point it remains unclear how much NO can escape scavenging by Hb and there is controversy regarding the concentration levels that are established in the SM. NO availability is determined by the balance between EC release and consumption by RBCs, unfortunately, both remain largely unidentified. A combination of modeling and experimentation investigates NO's fate in the vasculature. NO release rates were recorded from human umbilical vein ECs, loaded with an NO‐sensitive fluorescent probe and stimulated with acetylcholine. NO consumption rate was determined by flowing a NO gas stream on top of an RBC solution of specified hematocrit (Hct). Experiments suggest that the ECs can accomplish significant release rates (10−3nmole cm−2 s−1). The rate constant of NO consumption in porcine blood (kbl) at 45 % Hct was 3500 ± 700 s−1. The nonlinear dependence of kbl on Hct suggests a predominant role for extracellular diffusion in limiting NO uptake. These values combined suggest SM NO concentrations in the hundred nanomolar range.
The side‐effects after Taxotere® Treatment Affected by Polymorphism of Metabolic Enzymes in Taiwanese Breast Cancer Patients
Authors: Tsai Shih‐Meng , Lin Chiou‐Ya , Hou Ming‐Feng , Tsai Li‐Yu
Published: June 2021

Breast cancer has been the leading cause of death among Taiwanese women as well as women all over the world. Taxotere® has demonstrated the benefits versus other chemotherapy drugs. Nevertheless, there are different degrees of side effects with the chemotherapy medication. When these side effects occur, they must be cautiously treated, otherwise, they may be fatal. Therefore, this research is to explore the associations of genetic polymorphisms of metabolic enzymes and the side effects of Taxotere® in Taiwanese breast cancer. Breast cancer patients initiating Taxotere® treatment were genotyped for metabolic enzymes to identify variability of side effects. Fifty‐nine Taiwanese women treated for breast cancer were recruited. The polymorphisms of gene for metabolic enzymes were determined using restriction fragment length polymorphism of polymerase chain reaction products and the restriction enzymes. Based on this study, therefore, it is clinically beneficial for physicians to evaluate in advance the likelihood of the occurrence of side effects due to the application of Taxotere® by screening the genetic polymorphisms of their patients.
Cellulase assay as a method of monitoring grape ripening
Authors: Naleway John Joseph , Coleman Daniel John , Hawley Robin Michelle , Cook Gabriele Maria
Published: June 2021

Cellulases are an important family of enzymes found in many plant, bacterial, fungal, and yeast species. Plants encode cellulases [EC ] that catalyze the cleavage of the internal 1,4‐â‐glucosidic linkages of cellulose comprising their own tissue. It has been suggested that plant cellulases are essential for many facets of plant development, including abscission, fruit softening, vascular differentiation and symbiosis. A new fluorescent assay system has been developed to monitor cellulase enzyme activity in a continuous assay format at physiological pH values using the new substrate resorufin beta‐D‐cellobioside. Analysis of cellulase activity from grape samples obtained from the Pinot Noir and Pinot Gris varietals at developmental stages from before veraison through to the final ripe berry indicate the presence of a cellulase inhibitor or inhibitors whose level changes with ripening and correlates to the total mass ratio of dissolved sucrose (BRIX) and titratable acidity (TA) for the various samples. Protein and protease assays of the same samples appear to indicate that the inhibitor compound(s) may not be protein related. The identity of the inhibitor compound(s) is currently under investigation. These methods are being developed as a method to estimate optimum harvest conditions in an automated high‐throughput (HTS) format.
Retinoids Promote B Lymphopoiesis Even Under Sub‐optimal Conditions
Authors: Chen Xinrong , Kincade Paul W.
Published: June 2021

We recently found that all trans retinoic acid (ATRA) substantially accelerates progression in the B lymphoid lineage. Spleens of mice treated with ATRA had expanded populations of all major B cell subsets, while progenitor numbers in marrow declined. Retinoids acted directly on progenitors representing multiple stages of differentiation, and with cells from humans as well as from mice. Most of these experiments were done with highly purified progenitors in serum‐free, stromal cell‐free cultures optimized to generate CD19+ cells. Stromal cell co‐culture experiments were then initiated to learn if pDC differentiation was affected. Flt3 ligand (FL) was the only exogenous cytokine added in this circumstance, and we were surprised to find that CD19+B220+ lymphocytes emerged within two to three weeks of culture when 10−7 ATRA was present in the medium. All of the lymphocytes were Rag‐1+Mac1− and a portion were IgM+ B cells. B lymphopoiesis from adult progenitors is normally IL‐7 dependent, so experiments were conducted to determine if this cytokine might be present in our cultures. Indeed, no lymphocytes were generated when the IL‐7 specific M25 antibody was present, or when cultures were initiated with progenitors from IL‐7Rα knockout mice. Preliminary experiments suggest that ATRA slightly increases IL‐7 production in stromal cells, but levels must still be very low. We speculate that signals delivered from retinoid receptors reduce the stringency of those needed for B lymphopoiesis.

This work was supported by grants AI20069 and AI58162 from the National Institutes of Health.
Cleavage of the Notch Ligand Delta‐1 in the Regulation of T Cell Development
Authors: Gravano David Michael , Manilay Jennifer Oliveros
Published: June 2021

Signaling through the Notch ligand, Delta1 (DL1) facilitates normal thymocyte development. DL1 is presented to thymocytes by thymic epithelial cells (TECs) and can be proteolytically cleaved by ADAM proteases. However, the roles of DL1 cleavage products remain controversial. Mice lacking ADAM17 in TECs display blocks in T cell development, leading to speculation that Notch signaling between TECs and thymocytes is aberrant in these animals. However, this has yet to be experimentally tested. The objective of the current research is to determine whether ADAM17‐mediated cleavage of DL1 in TECs facilitates successful Notch signaling during thymocyte development. The OP9‐DL1 co‐culture system allows for the generation of mature T cells in vitro. Western blotting of OP9‐DL1 lysates with an intracellular Ab to DL1 showed steady state cleavage of DL1 and that the degree of cleavage is modulated by activators and inhibitors of ADAM proteases. Furthermore, co‐culture of thymocytes on OP9‐DL1 cells leads to an upregulation in DL1 cleavage. Non‐cleavable DL1 mutants and ADAM17 shRNA will be used to study the function of DL1 cleavage on thymocyte development in the OP9‐DL1 system. These studies will lend insight into the role of DL1 cleavage in the regulation of Notch signaling. Moreover, these data will provide evidence for whether processing of Delta1 is required for proper intrathymic cellular communication.
Senescent phenotype of CD8 T cells and correlation with metabolic status in nonagenarians
Authors: Chen Jian , Wu Qi , Hsu Hui‐Chen , Ravussin Eric , Scott Donald , Jazwinski Michal , Mountz John
Published: June 2021

Cellular metabolism is an important contributor to T cell functions. We hypothesized that a defined CD8 T cell phenotype would correlate with parameters of whole body metabolism in variously aged healthy subjects. T cells from 171 subjects who were young (21–35, n = 45), aged (60–89, n = 57), or nonagenarians (n = 63) were analyzed. The PHA‐induced proliferation on day 3 was assayed by 3H incorporation in the presence of either high concentration glutamine (10 mM) or glucose (10 mM). The best single phenotype that correlated with chronologic age was the percentage of CD28+Fas− CD8 T cells. This phenotype was used to define the immune age (IA) of an individual within the 3 age groups. To identify metabolic and hormonal factors that contributed to a younger IA, each age group was analyzed separately and also by sex. In contrast to nonagenarians, the aged group exhibited an uncontrolled proliferative response to PHA in the presence of glucose. The level of insulin, IGF and homocystine was correlated with IA for all age groups and the correlation was the highest for the aged female group (p<0.01). For the nonagenarians, the levels of growth hormone correlated with IA (p<0.001). For the aged group, IA most significant correlated with growth hormone and total daily energy expenditure (p<0.001) and DHEAS (p<0.001). For all groups, there are no correlation between IA and cholesterol, LDL, TG and CRP. We propose that genetic factors that regulate longevity and whole body metabolism also regulate T cell phenotype, including proliferation and glucose metabolism.