bluuebunny/crossref_metadata_2025 huggingface dataset:
Arcuate nucleus injection of anti‐insulin affibody prevents sympathetic response to circulating insulin
Authors: Luckett Brittany , Frielle Jennifer , Wolfgang Lawrence , Stocker Sean D.
Published: June 2021

Insulin contributes to cardiovascular dysfunction by central actions to increase sympathetic nerve activity (SNA). The arcuate nucleus (ARC) abundantly expresses insulin receptors, and direct ARC injection of insulin raises lumbar SNA (LSNA). Therefore, we hypothesized that peripheral insulin acts on ARC neurons to increase LSNA and tested this by using an anti‐insulin affibody to neutralize insulin. To test the efficacy of the affibody, anesthetized Sprague‐Dawley rats received bilateral ARC injection (40nL) of either an anti‐insulin (1μg) or control affibody (1μg) ~10 min before ARC injection of insulin (4μU). The anti‐insulin vs control affibody abolished the increased LSNA (120 min: 99±1% vs 128 ±8%, n=4; P<0.05). Blood pressure and heart rate were similar between groups. Second, ARC injection of anti‐insulin vs control affibody prevented the increased LSNA during a hyperinsulinemic‐euglycemic clamp (7.5mU/kg/min, IV; 114±5% vs 153±15%, n=5–6; P<0.05). Yet, ARC injection of gabazine elevated LSNA in both anti‐insulin vs control animals suggesting the affibody does not generally inhibit ARC neurons. These results indicate circulating insulin acts directly on ARC neurons to increase SNA.

Grant Funding Source : HL090826, APS UGSRF
Role of leptin and insulin on renal sympathetic nerve activity in high fat fed rabbits
Authors: Lim Kyungjoon , Burke Sandra L , Barzel Benjie , Armitage James A , Head Geoffery A
Published: June 2021

We determined the rapidity of changes in blood pressure (BP), heart rate (HR) and renal sympathetic nerve activity (RSNA) in response to a high fat diet using conscious rabbits and whether this involved insulin and leptin actions in the CNS. New Zealand White rabbits were implanted with renal electrode to measure RSNA. Rabbits were then placed on a normal or 13.5% high fat diet (HFD) for 1 or 3 weeks. Reflexes and stress responses were examined weekly. Rabbits were infused with peptide antagonists of Leptin (100μg) and Insulin (0.5U) intracerebroventricularly at week 1 and 3. After 1 week on the HFD, rabbits demonstrated a 4%, 8% and 30% greater BP, HR and RSNA, respectively (P<0.05). By the end of 3 weeks of HFD, BP, HR and RSNA were elevated by 10%, 5% and 82% (n=9). The insulin antagonist was most effective at 1 week in lowering BP, HR and RSNA while the leptin antagonist was most effective at 3 week of a HFD. The antagonists were minimally effective in control rabbits at these times. Sympathetic activation occurs rapidly within several days of a high fat diet initially associated with forebrain insulin signalling and later as fat is deposited, with forebrain leptin signalling. These changes appear to be associated with pathways mediating the sympathetic responses to acute emotional stress that are independent of baroreflex or chemoreflex pathways.
EEG brain wave band differentiation during a eustress state of humor associated mirthful laughter compared to a distress state
Authors: Berk Lee , Cavalcanti Paula , Bains Gurinder
Published: June 2021

Humor associated mirthful laughter, a novel eustress state, and a distress state differentially modulates neuroendocrine, immune, and psychological mood states. We used the 9 channel B‐Alert X10® EEG system with power spectral density (PSD), computed for left and right hemispheres, to determine if EEG metrics differentiated between a humor eustress state and a distress state. Eight video trial EEG sessions (states ‐ humor eustress vs. distress) were run on 2 individuals. Absolute PSD was used for the analysis of frontal theta (3–7Hz), alpha (8–12Hz), and beta (13–29Hz) of the left (F3) and right (F4) hemispheres. Using 10 sec epochs, ANOVA confirmed the humor state increased PSD across theta, p < .05; and alpha, p < .01. The opposite was true for beta where the eustress decreased PSD, p < .05. For differences between hemispheres, using 1‐way ANOVA, significant right‐left differences occurred across state for frontal theta, p < .05 and frontal alpha, p < .05. Significance was also found in the parietal for alpha, p < .05, but not for theta. PSD shows 1) both theta and alpha have greater left frontal (F3) EEG activity during the humor eustress state, which is associated with positive affect; and 2) right‐left hemispheric differentiation. In conclusion, PSDs computed for the left and right frontal hemispheres, using B‐Alert X10® EEG activity, differentiates a humor eustress state from a distress state.
Adenylyl Cyclase Inhibitors Reverse the Neurotoxic Effects of Manganese on Post‐Synaptic Dopamine D2 Receptors
Authors: Nelson Michael , Ojo Christiana , Adams Trevon , Carroll Margaret A , Catapane Edward J
Published: June 2021

Manganese (Mn) is a neurotoxin causing Manganism. It disrupts dopamine (DA) systems. The mechanism is not fully resolved. Gill lateral cilia of Crassostrea virginica are innervated by DA cilio‐inhibitory nerves. Previously we showed post‐synaptic DA receptors in gill lateral cells are D2 metabotrophic type, G protein‐coupled (Gai/o). Gai inhibits adenylyl cyclase (AC). Gâã opens K+ and closes Ca2+ channels. We showed Mn blocks DA post‐synaptic receptor. Here we observed membrane potentials of these cells of C. virginica with a fluorescent dye while measuring cilia beating rates. Applying HT to gill caused membrane depolarization and increased cilia beating rates. Applying DA after exciting cilia repolarized the membrane and decreased beating. Mn prevented the cilio‐inhibitory response and repolarization. Applying ATP or forskolin, an AC activator, to control or Mn treated gills increased beating without changing membrane potential. Applying MDL or SQ, AC inhibitors, to controls or Mn treated gills decreased beating without affecting membrane potential. The study shows correlation between membrane potential and cilia beating rates; that actions initiated by activation of D2 receptors can be differentiated to effects on AC and membrane potential; and neurotoxic effects of Mn can be overcome by AC inhibitors. This information is helpful to understand causes and treatments of Manganism.
Differential vascular reactivity of fetal and maternal placental arteries from melatonin treated nutrient‐restricted sheep to endothelium‐dependent and independent vasodilators
Authors: Shukla Praveen , Lemley Caleb O , O'Rourke Stephen , Meyer Allison M , Vonnahme Kimberly
Published: June 2021

Maternal nutrient restriction and decreased scotophase concentrations of melatonin have been associated with severely compromised pregnancies. We hypothesized that melatonin supplementation during a compromised pregnancy will improve the maternal (CAR) and fetal (COT) placental vascular reactivity thereby ensuring sufficient umbilical blood flow to the developing fetus. Pregnant ewes (n=32) were fed a control (ADQ) or nutrient restricted diet (RES) supplemented with (MEL) or without 5mg melatonin (CON) from day 50–130 of gestation. The sodium nitroprusside (SNP)‐induced relaxation of isolated CAR and COT arteries did not differ significantly between treatment groups. Independent of the maternal nutrition status, the sensitivity to bradykinin (BK) induced relaxation of COT but not CAR arteries, increased significantly (p<0.05) in MEL group. Moreover, CAR arteries showed increased sensitivity to BK‐induced relaxation, irrespective of the nutrition restriction, compared to COT arteries (pD2= 7.46 ± 0.1 CAR Vs 6.94 ± 0.2 COT). The norepinephrine and angiotensin II induced contraction of CAR and COT arteries did not differ significantly between treatment groups. The increased sensitivity of COT arteries to BK‐induced relaxation in MEL groups represents an important mechanism by which dietary melatonin supplementation improves the umbilical blood flow and placental nutrient transfer capacity.
Critical Role Of MCP‐1 In n‐6 Polyunsaturated Fatty Acids (PUFA)‐Induced Cardiac ER Stress In Diet‐Induced Obesity
Authors: GHOSH SANJOY , HALDER SWAGAT , BAHNIWAL RUPINDER
Published: June 2021

Pathways by which excess n‐6 PUFAs promotes cardiac inflammation and apoptosis is unclear. We predicted a role for ER stress in inducing such cardiotoxicity. Female C57/Bl6 mice were fed normal chow or high‐fat (40%) diets rich either in n‐6 PUFA or monounsaturated fatty acids (MUFA) for 5 weeks. Immunoblotting revealed upregulation of proteins like IRE‐1α, BiP, PERK and calnexin in the heart of n‐6 PUFA but not in MUFA fed hearts. Further, upregulation of CHOP‐1, caspase‐12, TUNEL and ubiquitinated protein expressions confirmed elevated apoptosis in n‐6 PUFA fed hearts. Interestingly, MCPIP [monocyte chemotactic protein‐1 (MCP‐1) induced protein], a nuclear transcription factor was also induced with n‐6 PUFA. As MCP‐1 is key in cardiac inflammation we hypothesized that n‐6 PUFA triggers cardiac ER stress in an MCP‐1 dependent manner. MCP‐1−/− mice hearts demonstrated significantly less ER stress and undetectable MCPIP levels in response to high n‐6 PUFA than MCP‐1+/+ hearts. Deletion of MCP‐1 also attenuated CHOP‐1, caspase‐12 and ubiquitinated proteins in response to n‐6 PUFA. These results indicate that dietary excess of n‐6 PUFA may lead to cardiac ER stress and apoptosis during diet induced obesity via MCP‐1 signaling. These data may provide an insight into possible mechanisms for cardiotoxicity of dietary oils like corn and sunflower oils in the western diet.
Characterization of Toll‐like Receptors and Beta‐defensin Expression in Porcine Glandular Epithelial Cells
Authors: Deachapunya Chatsri , Poonyachoti Sutthasinee , Kiatprasert Pongpat , Srisomboon Yosawee , Bauthong Norathee
Published: June 2021

Mucosal epithelial cells lining female reproductive tract (FRT) periodically expose to potential pathogenic organisms. It serves as a component of innate immunity response to invading pathogen through toll‐like receptors (TLRs) in a family of pattern‐recognition receptors (PRRs). Antimicrobial peptides such as porcine beta defensins (PBD1) and PBD2 is one of key effectors of pathogen and TLRs signaling mechanism in the respiratory and gastrointestinal systems to neutralize/control the progression of infection. In this study, we investigated the mRNA expression of TLRs and PBD in primary (PE‐I) and immortalized (IMM) porcine glandular epithelial cells which represented non‐cancer cell lines. TLRs mRNA expression in PE‐I and IMM cells was characterized by RT‐PCR. Both PE‐I and IMM cells showed the similar expression of all known TLRs and PBD but not TLR6, which were different from cancer cells. However, TLR2, 3, 4, 5 and 7 exhibited higher expression in estrogen‐deprived media. This evidence provides the first evidence of TLRs and beta‐defensin the antimicrobial peptide mRNA expression in normal porcine glandular epithelium. Functional relevance of TLRs mRNA expression and their functions based on cytokine production mediated by stimulation of pathogen‐associated molecular patterns (PAMPs) or microbial products could be further accessed by using this model.
Combined Analysis of Gene and MicroRNA Expression in Natural Killer Cells in Response to Exercise
Authors: Radom-Aizik Shlomit , Zaldivar Frank , Cooper Dan M
Published: June 2021

Natural Killers (NK) cells are unique innate immune cells that increase up to 5 fold in the circulating blood with brief exercise and are known to play a key role in first‐response defense against pathogens and cancer immunosurveillance. Whether exercise alters NK cell gene (GE) and microRNA (miRNA) expression is not known. 12 healthy men (20–29 y/o) performed 10, 2‐min bouts of cycle ergometer exercise interspersed with 1‐min rest at a constant work equivalent to □77% of V̇O2max). We used Affymetrix U133+2.0 arrays for GE and Agilent Human miRNA V2 Microarray for miRNAs. A stringent statistical approach (FDR<0.05) was used to determine that exercise significantly altered the expression of 986 genes, and 25 miRNAs. When we compared the GE changes that have been altered by exercise, with the miRNAs altered by exercise, we identified mainly pathways that are related to cancer and cell communication: p53 signaling pathway, Melanoma, Glioma, Prostate cancer, Adherens junction, and Focal adhesion, in which an interaction of miRNA and gene expression was plausible. Brief exercise alters GE and miRNA expression level in circulating NK cells in humans. These data support the hypothesis that exercise‐associated changes in NK miRNA expression play a role in NK gene expression in response to exercise stimulus, and ultimately, perhaps, in NK cell function in health and disease. Supported by NIH Grants P01HD048721 and UL1RR031985.
Various types of branching patterns of celiac trunk
Authors: srivastava ajai kumar , sehgal Garima , Sharma Pradeep Kumar , Kumar Navneet , Singh Ragini , Parihar Anit , Aga Pallavi
Published: June 2021

According to conventional anatomy, the celiac trunk divides into left gastric artery, common hepatic artery and splenic artery. The variations of branching patterns of celiac trunk may create surgical complications. Therefore the study has been carried out to identify and evaluate the spectrum of normal and variant anatomy of celiac axis by spiral computed tomography in the Departments of Anatomy and Radio‐diagnosis, C.S.M. Medical University, U.P., Lucknow, India. CT‐angiograms of 50 subjects were analyzed. In the present study, the celiac artery displayed classical trifurcation (28%), bifurcation (8%), quadrifurcation (36%), pentafurcation (20%) and hexafurcation (4%). Celiac trunk was absent in 4% cases. Knowledge of such variations can revolutionise successful surgical procedures around celiac trunk and the planning of image‐guided interventions.

No financial support is obtained from any source.
Morphometry of nerves responsible for sensory innervation of the hind limbs in rats
Authors: da Silva Letícia Oliveira Neri , de Aguiar Mendes Vânia Alice , de Amorim Milena Menezes , Sanada Luciana Sayuri , Fazan Valéria Paula Sassoli
Published: June 2021

The sensory innervation of the distal hind limb is achieved by branches of the sacral and lumbar plexuses. In rats, the skin of the dorsum of the foot is innervated mainly by the sural and saphenous nerves, with considerable overlapping between them. We aimed to compare the morphology and morphometry of the sural and saphenous nerves in young rats. Female Wistar rats (N=5) aged 30 days were perfused with 2.5% glutaraldehyde and had their right and left sural and saphenous nerves prepared for light microscopy and morphometry. Comparisons were made between segments (proximal and distal) and sides in the same nerves and between nerves of the same side. Both nerves are longitudinally symmetric on both sides. A lateral symmetry is also present on both nerves. In general, no differences were found between nerves, either for the fascicle or for the myelinated fibers morphometry. The similar number of myelinated fibers, associated with their similar size in both sural and saphenous nerves suggests that the innervation territory for both nerves is equally distributed. This information is useful for studies of sensory neuropathies, in particular, studies of pain.

Grant Funding Source : FAPESP, CNPq, CAPES and FAEPA
RICOM: a potential male contraceptive agent?
Authors: McNeil Rosie Thecla , Ekwere Ekwere Okon , Adebesin Abduljalil A , Okwuasaba Francis Kanayo , Ochigbo Adam John
Published: June 2021

The minor seed variety of RICOM – 1013‐J, a local contraceptive agent among the Rukuba women of Central Nigeria was studied.

The N‐hexane extract of RICOM has been shown to possess anti‐fertility properties in adult cyclic Sprague‐Dawley rats. The ether‐methanolic extract of the seeds has been proven to possess an oestrogen‐like effect on the female genital tract of adult Wistar rats. It induced vaginal opening in prenatal rat. However, the effect of RICOM on male fertility has not been documented.

Thus the aim of this study is to assess the effect of RICOM on testicular structure and function; semen parameters in vivo and in vitro and, to compare its effect to that of a known drug (diane35, which contains 0.035g cyproterone acetate and 2mg of ethinyl oestradiol/tab).

The semen parameters in vivo showed marked suppression of testicular function in both the groups treated with RICOM and diane35, when compared to that of the control.

We concluded that Ricinus communis‐linn suppresses testicular function and is a potential male contraceptive agent. This possibility should however be investigated further.
Chronic effect of parathyroid hormone on Na+/H+ exchanger isoform 3 gene proximal promoter
Authors: Neri Elida Adalgisa , Queiroz-Leite Gabriella Duarte , Bezerra Camila Nogueira Alves , Rebouças Nancy Amaral
Published: June 2021

Previous studies have shown that parathyroid hormone (PTH) chronically inhibits NHE3 in opossum kidney cells (OKP) by reducing total NHE3 protein and mRNA levels by modulation of the transcription. The purpose of this study was to elucidate the inhibitory mechanisms of PTH on the NHE3 gene promoter. Different segments of the proximal promoter (−157/+31; −85/+31; −65/+31 and −44/+31) were inserted in the pGL3‐ basic luciferase reporter vector, and point mutations were introduced in some regulatory elements, which were also evaluated by gel shift assays (GMSA). OKP cells were transiently transfect with the vector constructs and kept in serum‐free media for 24 h, then treated with 10−7M PTH for 24 h. PTH decreased the promoter activity of the fragments −65 and −44 (~21% and 31% respectively). Mutations on Sp1/Egr‐1 and AP2 elements completely abolished the PTH inhibitory effect. Inhibition of JAK/STAT pathway abolished the suppressor effect of PTH on fragment −65. In GMSA, PTH reduced the protein‐DNA affinity of the segment −44/+31.

In conclusion, these data suggest that the cis‐element(s) required for PTH responsiveness must be localized in the proximal promoter and the JAK/STAT pathway is involved in this inhibitory response.

Financial Support: FAPESP and CAPES
Investigation of the thermodynamics of DNA interactions with the LL‐37 peptide
Authors: Dodson CarolAnn Elizabeth , Zachary Ralph Karl , Craig Maria Lee
Published: June 2021

Previous studies of LL‐37, an antimicrobial peptide of innate immunity, show that LL‐37 has effects in lupus models and in breast and prostate cancer. This peptide is able to bind to DNA and diffuse through the lipid bi‐layer of cells, allowing entry of self‐DNA that can upregulate the immune system through Toll‐like receptor 9 (TLR9). In prostate and breast cancer, this small peptide becomes increasingly over‐expressed in tumor tissue as the cancer progresses. To further understand the biochemical nature of the DNA/LL‐37 complex, we have used electrophoretic mobility shift assays (EMSA) and isothermal titration calorimetry (ITC) to determine thermodynamic parameters, including ΔH, Kd, and peptide:DNA ratio, of complex formation between LL‐37 and CpG DNA sequences known to activate TLR9. By quantitative analysis of EMSA results, we observe a decrease in ethidium bromide staining when the concentration of LL‐37 is increased beyond that needed to shift all DNA and conclude that increasing LL‐37 concentrations leads to additional deposition of LL‐37 on the DNA. ITC results provide evidence of a stable complex between LL‐37 and CpG DNA. Future directions include studies of small molecules capable of blocking the binding of the peptide to the DNA. This work was supported by a Virginia Foundation of Independent Colleges Summer Undergraduate Research Fellowship.
Nucleotide‐Dependent Conformational Changes in the N‐Ethylmaleimide Sensitive Factor (NSF) and their Potential Role in SNARE Complex Disassembly
Authors: Moeller Arne , Zhao Chunxia , Fried Michael G. , Wilson-Kubalek Elizabeth M. , Carragher Bridget , Potter Clinton S. , Whiteheart Sidney W.
Published: June 2021

Homohexameric, N‐ethylmaleimide Sensitive Factor (NSF) disassembles Soluble NSF Attachment Protein Receptor (SNARE) complexes after membrane fusion, an essential step in vesicular trafficking. NSF contains three domains (NSF‐N, NSF‐D1, and NSF‐D2), each contributing to activity. We combined electron microscopic (EM) analysis, analytical ultracentrifugation (AU) and functional mutagenesis to visualize NSF's ATPase cycle. 3D density maps show that NSF‐D2 remains stable; whereas, NSF‐N undergoes large conformational changes. NSF‐Ns splay out perpendicular to the ADP‐bound hexamer and twist upwards upon ATP binding, producing a more compact structure. These conformations were confirmed by hydrodynamic, AU measurements. NSF‐ATP sediments faster with a lower frictional ratio (f/f0). Hydrodynamic analyses of NSF mutants define the structures underlying these conformational changes. Mapping mutations onto our 3D models allows interpretation of the domain movement and suggests a mechanism for NSF binding to and disassembly of SNARE complexes.

The electron microscopy and image analysis presented here was conducted at the National Resource for Automated Molecular Microscopy which is supported by the National Institutes of Health though the National Center for Research Resources’ P41 program (RR017573). The work was also supported by grants HL56652 and HL091893 from the National Institutes of Health to SWW. AM received support from the NIH Roadmap Award P50 GM073197.
SUMOylation of caveolin‐3: SUMO‐specific proteases SENP1 and SENP2 regulate deconjugation of SUMO‐3 and poly‐SUMO‐3 chains
Authors: Busija Anna Rebecca , Fuhs Stephen R. , Patel Hemal H. , Insel Paul A.
Published: June 2021

Mechanisms that regulate the interaction of caveolins with partners are poorly understood. We recently reported (JBC 286:14830–41 [2011]) the SUMOylation of caveolin‐3 by SUMO‐3, which is stimulated by expression of the SUMO E3 ligase PIASy and that SUMOylation influences interaction of caveolin‐3 with G‐protein‐coupled receptors. SUMO‐specific proteases (SENPs) deconjugate SUMO from targets and de‐polymerize poly‐SUMO chains. The seven human SENPs have distinct subcellular localizations as well as substrate and SUMO paralog specificity. We sought to determine if a specific SENP reverses SUMOylation of caveolin‐3. We screened SENPs for their ability to deconjugate SUMO‐3 from caveolin‐3 in vivo and found that expression of SENP1 or SENP2, but not SENPs 3, 5 or 6, dramatically reduced the accumulation of caveolin‐3‐SUMO‐3 conjugates. Proteins that localize to caveolae often have a caveolin‐binding motif (CBD, ΦxΦxxxxΦxxΦ: Φ = W, F or Y) that interacts with the caveolin scaffolding domain (CSD). We found that SENP1 has a CBD in its C‐terminal catalytic domain and SENP1 and SENP2 both co‐immunoprecipitate with caveolin‐3 in HEK293 cells. Taken together, these findings indicate that caveolin‐3 interacts with SENP1 and SENP2 and that SUMOylation of caveolin‐3 is dynamically regulated by the reciprocal enzymatic activities of PIASy and SENP1/SENP2. Research supported by NIH grants.
pH‐dependent Studies of E. coli Methylenetetrahydrofolate Reductase
Authors: Wang Jingjing , Trimmer Elizabeth E.
Published: June 2021

E. coli methylentetrahydrofolate reductase (MTHFR) is a flavoprotein that catalyzes the NADH‐ dependent reduction of 5,10‐methylenetetrahydrofolate (CH2‐H4folate) to 5‐ methyltetrahydrofolate (CH3‐H4folate). The pH‐dependency of MTHFR was studied in order to understand the roles of the ionizable active site amino acids in catalyzing the reaction. The reverse of the folate oxidative half‐reaction was studied at pH values 5–10 with the CH3‐H4folate‐menadione steady‐state kinetics assay. In this assay, menadione acts as an alternate electron accepter to oxidize the reduced enzyme. Our results show that a basic condition (pH 7–10) is helpful for the reverse folate reaction. An inflection point around pH 6 may reflect the pKa of His273, an amino acid implicated in a proposed proton transfer cascade necessary for folate deprotonation. Currently, we are characterizing the pH dependence of the NADH half‐reaction using the NADH‐menadione assay and the physiological reaction using the NADH‐CH2‐H4folate steady‐state assay. Research was supported by HHMI grant #52006298 to Grinnell College.
Iron contributes to hepatocyte insulin resistance in a cell model of nonalcoholic fatty liver disease
Authors: Messner Donald , Rhieu Byung Han , Godbout Daniel , Kowdley Kris V.
Published: June 2021

Increased body iron stores are associated with increased severity of nonalcoholic fatty liver disease (NAFLD). To clarify the role(s) of iron in NAFLD, this study investigated its effects on surrogate disease markers in AML12 hepatocytes: reactive oxygen species (ROS), stress kinase activation, and insulin resistance. Control AML12 cells defined baseline ROS levels (dichlorodihydro‐fluorescein fluorescence) and maximum insulin response (phospho‐activation of protein kinase B: EC50 near 1 nM insulin, maximum at 100 nM). Treatment with 50 μM iron (given with the lipophilic chelator 8‐hydroxyquinoline) increased ROS levels 8‐fold (p<0.01) and elevated phospho‐jnk and phospho‐p38. This was accompanied by a 33% decrease in response to 10 nM insulin (p<0.01). Under conditions in which fat accumulation alone had little or no effect on ROS levels or insulin response, cells pre‐loaded with stearic acid appeared sensitized to iron (ROS increased 18‐fold (p<0.01); insulin response decreased 51% vs. control), while cells treated with oleic acid appeared partially protected from iron (ROS levels increased 5‐fold (p<0.01); insulin response decreased 24%). We conclude NAFLD progression in this in vitro model is best recapitulated by the combined effects of saturated fat and iron. Studies to further define iron‐sensitive signaling pathways and evaluate novel therapies are justified. Supported by NIH grant AT03448.
NAD(P)H oxidase‐dpendent H2O2 production and RyR‐IP3R activation are required for insulin induced GLUT4 translocation and glucose uptake in skeletal muscle cells
Authors: Contreras-Ferrat Ariel , Vasquez Cesar , Espinosa Alejandra , Lavandero Sergio , Klip Amira , Jaimovich Enrique
Published: June 2021

Insulin‐regulated GLUT4 traffic has been widely explored, but the role of calcium and ROS signaling in this process is poorly understood.

We studied insulin‐induced GLUT4 traffic in GLUT4myc skeletal muscle cells.

Insulin‐induced exofacial exposure of myc epitope, glucose uptake and Akt activation were independent of extracellular calcium. The intracellular calcium chelator BAPTA‐AM inhibited Akt, p70S6K and ERK1 activation but not p‐ERK2. p‐ERK1/2 was inhibited by 2‐APB, an IP3R inhibitor. Cytosol directed parvalbumin reduced insulin‐dependent exposure of myc. Moreover, ryanodine and 2‐ APB partly inhibited myc exposure with an additive effect. The calcium ionophore ionomycin induced an increase in extracellular exposure of myc and was additive to insulin. In myotubes maintained in calcium‐free media, the effect of insulin was totally inhibited by ionomycin. Insulin increased H2O2 production measured with the cytosolic molecular sensor HyPer which was inhibited by NAC. p47 subunit of NADP(H)ox labeled differentiated myotubes. Both antioxidant agents, NAC and trolox partly reduced the externalization of myc as did the NADP(H)ox inhibitor apocynin and overexpression of catalase.

These data suggest that insulin induces an increase in NADP(H)ox‐dependent H2O2 production and RyR/IP3R calcium channels activation to foster GLUT4 translocation.

FONDAP 15010006, FONDECYT 3110170
Cell cholesterol levels determine glucose uptake and insulin resistance in skeletal muscle
Authors: Llanos Paola , Contreras-Ferrat Ariel , Osorio-Fuentealba Cesar , Espinosa Alejandra , Hidalgo Cecilia , Jaimovich Enrique
Published: June 2021

In skeletal muscle, cholesterol is enriched in the transverse tubule membranes (TT), where insulin‐promoted GLUT4 translocation occurs. To test the role of TT cholesterol content, we fed mice a high fat diet (HFD) and measured insulin triggered glucose uptake. Cholesterol levels were measured in TT‐enriched triad fractions isolated from skeletal muscle of male C57BL/6J mice fed either a normal or a HFD (60% fat) for 8 weeks. Isolated muscle fibers from flexor digitorum brevis were used for fluorescence glucose uptake determinations. Akt activity in myotubes was determined by Western blot analysis. Relative to controls, body weight, body fat content and metabolic variables including insulin resistance were higher in HFD fed mice. Cholesterol levels in triads from HFD‐fed mice were 30% higher than in controls. Preincubation of myotubes with Methyl‐β‐cyclodextrin to remove cholesterol partially inhibited Akt phosphorylation in controls, and increased both basal and insulin‐induced glucose uptake in muscle fibers from controls or HFD‐fed mice. Cholesterol removal restored insulin sensitivity in HFD‐fed mice, independently of Akt and CaMKII inhibition. Our findings indicate that cholesterol removal from muscle fibers may be a strategy for improving insulin resistance in HFD‐fed mice and suggest that TT cholesterol has a physiological role in glucose transport.

FONDECYT 3110105, FONDAP 15010006.
myo‐Inositol oxygenase identified in Drosophila melanogaster
Authors: Jones Melissa Kaye , Eldon Elizabeth D. , Klig Lisa S.
Published: June 2021

myo‐Inositol oxygenase (MIOX) catalyzes the first committed step in eukaryotic inositol catabolism. In human diabetics, inositol homeostasis is often disturbed through mechanisms that are largely unknown. MIOX is expressed in the kidneys and in extra‐renal tissues where diabetic complications occur. MIOX has not been studied in the model organism Drosophila melanogaster. Establishing that a gene for MIOX exists in D. melanogaster allows for studies of this organism to have cross‐species relevance to humans. The protein encoded by the D. melanogaster CG6910 gene has 54.6% identity to the Mus musculus MIOX protein. CG6910 encodes a protein containing the cognate hairpin loop structure, iron‐binding aspartic acid residue, and PKC binding sites found in mouse and human MIOX. Most of the PKA binding sites are also conserved and novel sites have been found. Flies with CG6910 RNAi ubiquitously activated by an Act5C‐GAL4 driver, similar to carbon/energy starved flies, did not survive more than five days with inositol as the sole carbon/energy source. Western blot analyses reveal decreased MIOX expression in RNAi flies with the Act5C‐GAL4 driver. Other preliminary experiments include a biochemical assay demonstrating MIOX activity in D. melanogaster. These studies indicate that CG6910 encodes MIOX in D. melanogaster and may contribute to understanding the role of inositol catabolism in development and diabetes.
Refinement of virtual screening binding criteria for PPARγ therapeutic screening
Authors: Lewis Stephanie Nicole , Brannan Lera , Bassaganya-Riera Josep , Bevan David
Published: June 2021

In an effort to find more effective therapeutics for treating chronic inflammation‐related diseases, such as type 2 diabetes and inflammatory bowel disease, we used computational and experimental methods to search for novel agonists directed at peroxisome proliferator‐activated receptor‐gamma (PPARγ). Molecular docking and dynamics techniques were used to carefully characterize PPARγ as a target for treating these types of diseases. Specifically, we coupled steered molecular dynamics techniques with detailed crystal structure analysis to improve the hit prediction of high‐throughput virtual screening. We have predicted which residues within the binding cavity of PPARγ are specific to full agonist and partial agonist binding. For example, residues on helix 3 near the middle of the binding cavity (i.e., Arg288) play roles in ligand binding and possibly ligand type distinction. The ligand type‐specific residue lists suggest interactions necessary for binding, which are then applied to increase success in identifying potential novel agonists via docking. We also generated ligand‐based pharmacophores, which, coupled with the residue information, will aid in more successful and faster virtual screening for compounds that will activate PPARγ. NIH‐NIDDK grant 1F31DK091186‐01A1 awarded to DRB and SNL.
Altered GIPC expression: potential role in cancer progression
Authors: Reed Brent , Cefalu Chris , Mastrodomenico Matthew , Campbell Taylor , Alexander Jonathan Steven , Williams Briana Jill , Huang Shile , De Benedetti Arrigo , Li Yan , Lamb-Hall Maria
Published: June 2021

GIPC is an adapter protein that couples the proper trafficking of numerous proteins affecting cancer progression to the movement of the myosin VI motor protein. GIPC is elevated in human prostate cancer tissue and suggests subsequent disruption of proper protein targeting could contribute to cancer progression. This study assesses in vitro the ability of targeted suppression of GIPC to block known steps in prostate cancer progression. Lentiviral shRNA suppression of GIPC in DU145 cells had no effect on cell growth or invasion, but reduced IGF‐1 mediated cell migration as well as adhesion to endothelial monolayers and subsequent transmigration. Cell surface levels of E‐cadherin, GLUT1, and IGF‐1 receptor were respectively elevated, reduced, and unaltered. Lowered expression of GIPC in endothelial monolayers, however, increased DU145 adhesion and transmigration and reduced tight junction integrity. Thus, targeting GIPC in prostate cancer cells could potentially block several steps in cancer progression, but mis‐targeted reduction of GIPC in endothelial cells distal to the tumor could enhance inflammatory processes by compromising tight junction integrity while increasing metastatic potential for circulating tumor cells to adhere and transmigrate though compromised endothelial barriers. (Funding: Feist‐Weiller Cancer Center, DOD PCRP, AHA, ADA, and Malcolm Feist Cardiovascular Research Endowment)
A novel testis‐specific isoform of the protein phosphatase1 inhibitor I‐2 (Ppp1r2) is expressed in developing germ cells during sperm morphogenesis.
Authors: Ramdas Shandilya , Sinha Nilam , Vijayaraghavan Srinivasan
Published: June 2021

The protein Inhibitor‐2 (I‐2, PPP1R2) of the serine/threonine phosphatase PP1 is one of the ancient PP1 binding proteins. An isoform of PP1, PP1γ2, is highly expressed in testis. This isoform, present only in mammals, is essential for spermatogenesis and sperm function. The coding sequence and the protein product of this message were not identified. We now show that this 0.9 kb message is expressed in developing germ cells during differentiation of haploid spermatids into spermatozoa. Using a spermatid cDNA library and RT‐PCR of testis RNA we have shown that the 0.9 kb mRNA arises due to retention of the last intron (intron 4) that is spliced out in the somatic form of I‐2 mRNA. This intron retention leads to the truncated message that generates a protein with a unique C‐terminus not found in the somatic cell form of I‐2. Protein sequence analysis using MS of heat stable testis extracts confirmed the amino acid sequence of the unique germ cell form of I‐2. Remarkably two other protein regulators of sperm PP1γ2, sds22 and I‐3, are also expressed as testis specific isoforms during late stages of spermatogenesis. Studies are underway to determine the significance of high levels of expression of testis specific versions of these PP1 regulators in spermatogenesis and sperm function. (NIH HD38520)
Regulation of bone remodeling by glycogen synthase kinase 3β in a transgenic mouse model
Authors: Park Duri , Ko Ryeojin , Lee Soo young
Published: June 2021

Glycogen synthase kinase 3β (GSK3β) plays roles in cell proliferation and differentiation. Numerous studies demonstrated roles for GSK3β in osteoblast differentiation. However, the role by which GSK3β regulates osteoclast fate remains unclear. We investigated the role of the GSK3β in osteoclast differentiation using transgenic mice overexpressing GSK3β specifically in the osteoclast lineage. We found that the transgenic mice resulted in increasing bone density or inhibition of osteoclastogenesis in vivo and in vitro. Importantly, osteoclast differentiation and induction of NFATc1 expression or localization to nucleus by RANKL treatment were profoundly diminished in transgenic mice. Our study demonstrates a novel role for GSK3β as a negative regulator of osteoclastogenesis.
Cysteine‐rich protein 2 regulates vascular smooth muscle cell migration via influencing p130Cas localization
Authors: Chen Chung-Huang , Hsieh Chia-Chu , Chuang Yung-Jen , Yet Shaw-Fang
Published: June 2021

Cysteine‐rich protein (CRP) 2, expressed in vascular smooth muscle cells (VSMCs) of blood vessels, plays a critical role in regulating VSMC migration and vascular remodeling. To investigate the molecular mechanisms by which CRP2 regulates migration, we first determined subcellular localization of CRP2. Transfection of VSMCs with CRP2‐EGFP constructs revealed that CRP2 is associated with actin cytoskeleton, suggesting a cytoskeletal function of CRP2. CRP2 null VSMCs adhered to or spread on extracellular matrix normally. In response to chemoattractant stimulation, lack of CRP2 increased VSMC lamellipodia formation. Immunofluorescence staining revealed that CRP2 colocalized with phospho‐p130Cas, a scaffold protein important for lamellipodia formation, at focal adhesions (FAs) at the terminal ends of stress fibers in non‐migrating cells. Mammalian 2‐hybrid and coimmunoprecipitation assays further confirmed the interaction of CRP2 and p130Cas. Interestingly, in migrating cells phospho‐p130Cas was present at the leading edge of lamellipodia and FAs while CRP2 was only found at FAs. Taken together, our results suggest that CRP2 may sequester p130Cas at FAs, thereby reducing lamellipodia formation and subsequent cellular migration. This work was supported in part by National Health Research Institutes (Taiwan) Grant CS‐100‐PP‐04 and National Science Council (Taiwan) Grant 98‐2320‐B‐400‐004‐MY3.
AMPK Regulates Lipolysis by Phosphorylation of Desnutrin/ATGL at Serine406
Authors: Abbott Marcia J , Sul Hei Sook
Published: June 2021

Desnutrin has been identified as the major triacylglycerol (TAG) lipase. However, very little is known about its direct activators. Phosphorylation sites have been identified in desnutrin, serine 406 and 430. We aimed to establish the regulatory activity of these residues in vitro and in vivo and to ask if AMPK is the kinase that phosphorylates these sites. Our data show that AMPK phosphorylates desnutrin at S406 but not S430 in vitro. HEK 293 cells were transfected with desnutrin or alanine mutants to prevent phosphorylation at S406, S430, or both. Glycerol release was measured after AICAR treatment, a potent AMPK activator. AICAR resulted in an increase in glycerol release in desnutrin overexpressing cells. S406 mutation (S406A) resulted in a reduction in glycerol release with control and AICAR cells. These data indicate that S406 is specifically required for desnutrin activity. To determine the physiological relevance in adipocytes, differentiated 3T3ΔCAR adipocytes were infected with desnutrin or S406A. AICAR and Metformin increased FA release over control cells. S406A decreased FA release with AICAR and Metformin treatments. To determine the consequence of S406 in vivo, desnutrin and S406A were introduced by adenovirus. S406A resulted in a decreased in vitro lipase activity and increased TAG. This study provides evidence for direct regulation of desnutrin activity, in vitro and in vivo, at S406 via AMPK.
Glycosomal localization of dihydroxyacetone phosphate acyltransferase LmDAT is important for lipophosphoglycan biosynthesis
Authors: Patel Nipul , Zhu Tongtong , Dhalladoo Subbhalakshmi , Zufferey Rachel
Published: June 2021

Protozoan parasites of the genus Leishmania are the causative agents of several human and animal diseases collectively called leishmaniases. The parasite cycles between an insect vector the sand fly, and mammalian host's macrophages. Cell‐surface components protect the parasite from the host immune system and constitute virulence factors. Ether glycerolipids are a significant part of the parasite's membranes and ether glycerolipid based macromolecules are also important virulence factors. Thus, enzymes involved in synthesis of ether lipids in Leshmania may represent a drug target for control of the disease. In L. major, a glycosomal dihydroxyacetonephosphate acyltransferase, LmDAT, initiates the ether lipid biosynthetic pathway. We demonstrated that an active LmDAT is critical for normal growth and survival of the parasite during stationary phase. Deletion of the N‐terminal amino acid residues of the enzyme reveals that this domain may be important for proper folding of the enzyme and hence for its activity. Abrogation of its C‐terminal glycosomal targeting sequence leads to its extraglycosomal localization. However, this mislocalization does not affect its enzymatic activity but alters the synthesis of the ether‐lipid based virulence factor, lipophosphopglycan, demonstrating that glycosomal subcellular localization is critical for the production of ether‐lipids.

This work was funded by American Heart Association.
Is the eukaryotic cis‐prenyltransferase a heteromer? The role of NgBR and its yeast ortholog Nus1 in protein glycosylation
Authors: Grabinska Kariona A , Guan Ziqiang , Sessa William C
Published: June 2021

Dolichyl phosphate (Dol‐P) serves as an obligate lipid carrier in different type of protein glycosylations. Dol‐P precursor ‐ polyprenyl pyrophosphate ‐ is synthesized by a cis‐prenyltransferase (cisPTase) activity. Recently we have shown that NgBR is as an essential component of the Dol‐P biosynthetic machinery. Loss of NgBR results in deficit of cisPTase activity, leading to diminished levels of dolichol‐linked oligosaccharides and a reduction in protein N‐glycosylation. NgBR interacts with the previously identified cisPTase hCIT, enhances hCIT protein stability, and promotes Dol‐P synthesis. Inactivation of yeast ortholog of NgBR, Nus1 gives lethal phenotype and causes N‐glycosylation defect. In this work we confirmed that shutting down expression of NUS1 results in N‐glycosylation defect and demonstrated that this phenotype is related to loss of cisPTase activity and dolichol synthesis. Glycosylation‐dependent phenotypes are complemented by expression of bacterial type undecaprenyl pyrophosphate synthase. We hypothesize that active eukaryotic cisPTase is not a homodimer as crystallized bacterial enzyme but heteromer consisting of: NgBR and hCIT in humans or Nus1 and Rer2 in ER and Nus1 and Srt1 in lipids bodies of yeast S. cerevisiae. Reconstitution of active eukaryotic cisPTase is in progress.

This work was supported by grant R01HL081190 from the NIH to WCS
Attachment capabilities of erythroleukemia cells to normal human fibroblasts
Authors: Barnes Carol , Birckbichler Paul J
Published: June 2021

Erythroleukemia cell lines, K562 and HEL, show a differential response to tissue transglutaminase (tTg) activation following exposure to retinoic acid. To study a role for tTg in cell‐¬cell attachment both cell lines were independently added to preconfluent and confluent normal human fibroblasts (WI‐38). No cell attachment was evident 6 hours after 175,000 K562 cells were added to adherent WI‐38 cells. After 12 hours small numbers of single cells adhered to the WI‐38 cells. Cell attachment continued through 48 hours with colonies evident as large as 3 to 5 cells with the greatest attachment occurring in the confluent cultures. To monitor cell growth during the 48 hour period, a control of K562 cells identical to the experimental flask was set up. The suspended cells in the experimental cultures were routinely 80 to 90% of those in the control K562 culture, indicating 10 to 20% of the K562 cells at any given time were attached to the WI‐38 cells. The attachment was sufficiently strong such that manual agitation failed to remove the attached cells. These observations suggest that a stabilized extracellular matrix played a role in the attachment. The failure to remove all the attached cells by agitation raises the possibility of a covalent linkage (bridge) between the WI‐38 and K562 cells; possibly a disulfide and/or isopeptide bond(s). Support from College of Health, Environment and Science and Slippery Rock University.
Mechanisms of resistance to quaternary ammonium compounds and other antimicrobials in Salmonella enterica
Authors: Stamm Sarah , Herson Diane
Published: June 2021

Each year nearly 1.4 million people in the United States are infected with Salmonella as a result of food poisoning. Antimicrobials are used for both the prevention and treatment of salmonellosis, and a major concern is that resistance to these agents will develop. Salmonella enterica Strains with Reduced Susceptibility (SRS) to Quaternary Ammonium Compounds (QAC) were generated by repeatedly passaging strains in increasing concentrations until a Minimum Inhibitory Concentration (MIC) of 400 ppm was obtained. DSRS strains were generated by culturing SRS stains in the absence of the agent until their MIC returned to that of the parent strain. Parent, SRS, and DSRS strains were tested for cross resistance to four antibiotics. Strains were then tested for efflux pump activity by qPCR analysis of the acrB and marA genes. Unlike DSRS, SRS strains showed cross resistance to all four antibiotics. These strains showed upregulation of the acrB gene while DSRS showed expression similar to the parent strains. For marA gene expression, both SRS and DSRS strains showed upregulation compared to the parent strain. This project was funded by Howard Hughes Medical Institute.
Control of Smoke‐Induced Proliferation and Migration of Human Breast Cancer Cells by PK11195, a Translocator Protein (TSPO) Antagonist
Authors: Mukherjee Shyamali , Das Salil K
Published: June 2021

Cigarette smoking is linked to breast cancer, particularly in young women. However, the exact mechanism is unknown. We have recently established that TSPO is an important biomarker for breast cancer; its expression is increased with an increase in aggressiveness of cancer. The present study investigates whether exposure of mainstream (MS) as well as Sidestream (SS) cigarette smoke induces TSPO‐mediated cell proliferation and migration in a variety of human breast cancer epithelial cell lines and if so whether PK11195, a TSPO antagonist can counteract these effects. Human breast cancer cell lines (MCF10, MCF 12A and MCF 12F) were grown in DMEM/F‐12 and exposed to either MS or SS smoke generated from 1R3F cigarettes in a THRI MS‐SS smoke exposure machine using the modified version of CULTEX cell culture smoke exposure system. A sham control (SH) group was also used where cells were exposed to the same environmental condition without smoke exposure. Induction of cell proliferation and migration was observed at low doses of smoke whereas cellular apoptosis was observed at high doses in all cell lines regardless of MS and SS exposure. A combination of immunohistochemical, Western blot and receptor binding studies revealed an over‐expression of TSPO in all cell lines by both MS and SS smoke. Furthermore, both MS and SS smoke caused a higher level of TSPO in nuclei associated with an increase in nuclear cholesterol transport. The proliferation and migration was controlled when treated with PK11195, suggesting a therapeutic potential of PK11195 in controlling smoke induced breast cancer development. ( Supported by FAMRI Clinical Innovator Award # 062415).