bluuebunny/crossref_metadata_2025 huggingface dataset:
Three days of unloading induces pre‐translational changes in human skeletal muscle
Authors: Gustafsson Thomas , Österlund Ted , von Walden Ferdinand , Trappe Todd , Linnehan Rick , Tesch Per
Published: June 2021

Any change in skeletal muscle mass is controlled by the balance of protein synthesis and degradation. These processes require signalling through the PI3K‐Akt‐mTOR and ubiquitin‐proteasome pathways. This study explored whether these pathways are activated soon after withdrawal of weight bearing. Muscle biopsies were obtained from m. vastus lateralis and m. soleus, of eight healthy men prior to and following three days unilateral lower limb suspension (ULLS). mRNA levels of IGF1, Caspase‐3, Calpain‐2, Foxo1A, Foxo3A, Atrogin‐1, MuRF‐1, 4E‐BP1, and Myostatin were quantified in combination with protein measurements of Akt, Foxo 1 and 3, p38 and mTOR. Caspase‐3, Calpain‐2, Atrogin‐1, MuRF‐1 and 4E‐BP1 mRNA increased after ULLS in m vastus lateralis but not in m soleus. Myostatin increased in both muscles and to a greater extent in m. soleus. Total amounts and phosphorylation of Akt, Foxo1 and 3, p38 and mTOR were unaltered (p>0.05). These results suggest that Caspase‐3, Calpain‐2, Atrogin1, MuRF1, 4E‐BP1 and Myostatin are pretranslational regulated in human skeletal muscle within three days of ULLS. Given that neither of measured signalling pathways was altered, it appears that other pathways stimulated the transcription of Atrogin1 and MuRF1. The difference in the gene expression pattern across m soleus and m vastus lateralis muscles indicates the atrophy mechanism may be different in the two muscle types.
Acetaminophen and oxidant‐induced osteopontin in the myocardium
Authors: Jaques Kathryn M , Denhardt David T , Merrill Gary F
Published: June 2021

Acetaminophen (APAP) exhibits cardioprotective, antioxidant effects in the injured mammalian myocardium. A number of mechanisms mediate ischemia/reperfusion‐induced myocardial injury, the most prevalent involving oxidative stress. The oxidant scavenging properties of APAP appear to terminate many signal transduction cascades which would normally become activated by injury‐generated reactive oxygen species (ROS). ROS target substrates of cell metabolism, growth, chemotaxis, survival, and death. One such substrate is the cytokine osteopontin (OPN), a phosphoglycoprotein found in body fluids and extracellular matrix of mineralized tissues. Its expression is minimal in normal adult cardiomyocytes and fibroblasts, but increases during pathological events including tissue remodeling. A recent study found that in OPN‐deficient mice the myocardial angiogenic response and post‐infarct tissue remodeling were reduced relative to wild type. These observations prompted us to hypothesize that APAP will reduce oxidant‐induced OPN expression in dogs. Respiratory, metabolic, and hemodynamic variables were measured in anesthetized, open‐chest dogs after administration of H2O2, in the presence or absence of 15mg/kg APAP. Femoral plasma was analyzed for OPN levels using western blot and ELISA. Preliminary results suggest APAP‐treated dogs express lower OPN levels than vehicle‐treated dogs.
Regulation of NADPH oxidase 5 activity by PKC and MAPK pathways.
Authors: Pandey Deepesh , Fulton David
Published: June 2021

NADPH oxidase 5 (Nox5) is a unique Nox isoform in that its activity is independent of the protein:protein interactions that coordinate the activation of other Nox isoforms and is instead regulated by cytosolic Ca2+ levels. Previously, we showed that PMA induces the sustained activation of Nox5 that was dependent on the phosphorylation of specific serine and threonine residues which control the calcium‐sensitivity of the enzyme. However, the specific kinase(s) mediating the phosphorylation of Nox5 are not known. We hypothesize that Nox5 is activated by the coordinated phosphorylation of MAPK and PKC pathways and that different isoforms of PKC are required for site‐specific phosphorylation. COS‐7 cells expressing Nox5 were incubated with the PKC inhibitor LY379196. LY379196 significantly attenuated PMA‐dependent increases in Nox5 activity and reduced the phosphorylation of S498 and to a lesser extent T494. These results indicate that PKC‐beta or a similar isoform participates in the phosphorylation and activation of Nox5. In addition to activation of PKC, PMA robustly stimulated the phosphorylation of Erk1/2. Furthermore, inhibition of Mek1 using PD98059 and U0126 significantly reduced the phosphorylation and activity of Nox5 in response to PMA. Future studies will employ a genetic approach to identify whether Erk or PKC isoforms directly phosphorylate and activate Nox5.
Characterization of an Epilepsy‐associated variant of the AE3 Cl‐/HCO3‐ exchanger
Authors: Vilas Gonzalo Luis , Freund Paul R. , Casey Joseph R.
Published: June 2021

Anion exchanger 3 (AE3), expressed in brain, heart and retina, extrudes intracellular HCO3‐ in exchange for extracellular Cl‐. There are two isoforms of AE3, brain or full length AE3 (AE3fl) and cardiac AE3 (cAE3). Epilepsy is a heterogeneous group of disorders characterized by recurrent unprovoked seizures that affect about 50 million people worldwide. Idiopathic generalized epilepsy (IGE) accounts for approximately 30% of all epilepsies. Interestingly, the occurrence of the A867D AE3 allele in humans has been reported to be associated with the development of IGE. Moreover, AE3 knockout mice are reported to display a reduced threshold for chemically induced seizures. We evaluated wild type (WT) and A867D AE3fl functional and physiological parameters in transfected HEK cells. Although both proteins are processed similarly to the plasma membrane (32 ± 2 % vs 37 ± 5 %, respectively), A867D AE3fl displays a significantly reduced transport activity (47 ± 13 %, p < 0.05) when compared to the WT. Protein Kinase A (PKA) regulates the transport activity of both proteins since stimulation with 8‐Br cAMP increases transport and pre‐incubation with the PKA‐specific inhibitor H89 abolished this increase. Together this suggests that the decreased activity of A867D AE3 may cause changes in cell volume and abnormal intracellular pH. In the brain, these alterations may promote neuron hyperexitability and the generation of seizures. This research is supported by Canadian Institutes of Health Research.
pH sensor in the kidney
Authors: Sun Xuming , Petrovic Snezana
Published: June 2021

Proton receptors are G‐protein‐coupled receptors, which can sense pH in the physiological pH range. One of the receptors, GPR4, is expressed at high levels in kidneys, overexpressed in kidney tumors, and mediates pH‐dependent increase of intracellular cAMP. We used cultured outer medullary collecting duct cells, which express GPR4, and GPR4 knockout mice (KOs) (Yang et al., Mol Cell Biol, 2006) to examine if GPR4 is involved in acid‐base regulation. Acid secretion in OMCD cells was measured with NH4Cl−prepulse and pH sensitive dye BCECF‐AM as pH recovery from intracellular acidification. Control cells increase acid secretion if incubated in media of low pH (7.0), whereas cells transfected with anti‐GPR4 synthetic siRNA do not. Cells transfected with control siRNA were not different from the control, non‐transfected cells. GPR4 KOs have mild metabolic acidosis (arterial blood HCO3−of 21.2 ± 1.5 vs. 24.9 ± 0.62 mM in WTs; pCO2 of 34 ± 2.6 vs. 38 ± 1.1 mmHg in WTs, and slightly higher Cl− concentration). After acid loading (280 mM NH4Cl for 5 days), both WTs and GPR4 KOs were acidotic, but WTs decreased urine pH from 6.0 ± 0.08 to 5.5 ± 0.05 whereas urine pH of GPR4 KOs did not change (6.1 ± 0.05 vs. 6.0 ± 0.1). mRNA expression of other pH sensing receptors, OGR1 and TDAG8, examined with real‐time RT‐PCR, was not increased in the kidneys of GPR4 KOs. We also examined the expression of unrelated but exquisitely pH sensitive calcium receptor (CaR). CaR expression was increased in the kidneys of GPR4 KOs at baseline (60%) and after acid loading (100%) compared to WTs. Our in vitro and in vivo data suggest that GPR4 mediates adaptive response to acidosis and that it may act in concert with CaR, a possible explanation for only mild acidosis in GPR4 KOs.
Detection of 3rd mechanism in renal blood flow via high‐resolution, time‐varying spectral analysis of records subjected to chirp forcing of blood pressure
Authors: Siu Kin , Lau Catherine , Cupples William , Chon Ki
Published: June 2021

It is generally agreed that renal autoregulation is mediated by myogenic (0.01–0.3 Hz) and tubuloglomerular feedback (0.02–0.05 Hz) mechanisms. Recently a potential 3rd mechanism that operates at ~0.01 Hz has been reported based upon step response experiments. To begin its characterization, 6 anesthetized, normotensive rats were instrumented for blood pressure (BP) and renal blood flow (RBF) measurements. We forced BP with linearly increasing frequencies (frequency range 0.001 – 0.02 Hz, amplitude ± 7 mmHg). The motivation for using linearly increasing frequencies in BP forcing is that if such a third mechanism exists, it should resonate at its characteristic frequency in the resultant RBF recordings and that RBF power at that frequency should be amplified beyond that in BP. Comparison between the time‐varying spectral amplitudes of BP versus RBF shows that when the BP forcing frequency passed ~0.01 Hz, there was greater spectral amplitude at the same frequency in RBF than in BP. The significance of this increase of spectral amplitude in RBF was verified by testing against white noise signals. Any spectral power above white noise spectral amplitude suggests that such oscillation does exist and not a consequence of some random occurrence. We conclude that a third mechanism contributes to autoregulation of RBF and that it resonates at ~0.01 Hz. Funded by CIHR & NHLBI
Evaluation of hemodynamic renal in the isolated perfused rat kidney after nitric oxide blockade
Authors: Fiorino Patricia , Farah Vera Azevedo , Darini Kalebe G , Araujo Iara Cristina , Leite Ana Paula Oliveira , Fonteles Manasses Claudino
Published: June 2021

Chronic administration of the nitric oxide synthase inhibitor L‐NAME causes arterial hypertension and renal injury. The purpose of this study was to evaluate in hypertensive animals induced by L‐NAME the renal hemodynamic and renal function using isolated perfused kidney model. The rats (n=5, each group) were divided in: 1)Normotensive (Nt), without treatment and 2)Hypertensive group: L‐NAME administration in the drinking water (600 mg/L) during 7 days (H7). L‐NAME treatment induces an increase in arterial blood pressure (143 +/− 2.2 vs. 105+/− 1.8 mmHg, H7 vs. Nt). Isolated kidneys were perfused with Krebs‐Henseleit solution containing 6g of previously dialysed bovine serum albumin for 60 min. The treatment increased the perfusion pressure (Nt=103.4 +/−1.2; H7=143+/−7.66 mmHg), and renal vascular resistance (Nt=4.5+/−0.51; H7=14.01+/−0.7 mmHg). These alterations were accompanied by a reduction of the urinary flow (Nt=0.16+/−0.01; H7=0.09+/−0.02 mL g(‐1) min(‐1)) and glomerular filtration rate (Nt=0.84+/−0.075; H7=0.19+/−0.032 mL g(‐1) min(‐1)). These finding showed that NO generated within the kidney contributes to the regulation of renal vascular tone in rats. In conclusion, our data suggests that NO inhibition induces dramatic changes in renal function demonstrated in the ex‐vivo kidney perfusion.

Financed by CNPq and MackPesquisa, Brazil
Acute heme oxygenase inhibition augments afferent arteriolar autoregulatory constrictor responses in angiotensin II‐hypertensive rats
Authors: Botros Fady T. , Navar L. Gabriel
Published: June 2021

Heme oxygenases (HO‐1; HO‐2) catalyze conversion of heme to free iron, carbon monoxide (CO), and biliverdin/bilirubin and exogenous CO dilates afferent arterioles (AA). Because chronic angiotensin II (Ang II) infusion induces renal HO‐1 expression, we determined if HO modulated AA autoregulatory responsiveness in Ang II‐dependent hypertension. Rats were infused with Ang II (80 ng/min for 11 to 13 days). The blood‐perfused juxtamedullary nephron preparation was used to measure AA diameter responses to increases in renal perfusion pressure (RPP) before and during superfusion with 15 μM Chromium mesoporphyrin (CrMP) to inhibit HO. Systolic blood pressure significantly increased from 129±6 mmHg to 188±7 mmHg 10 days after Ang II infusion, n=7, p<0.05. Increases in RPP from 100 to 125 and 150 mmHg significantly reduced AA diameters by 5.1±1.2% and 9.5±2.2%, n=6, p<0.05. Acute HO inhibition did not alter basal diameters at 100 mmHg (18.0±0.6 μm vs. 18.5±1.0 μm, n=6), but AA constrictor responses to increases in RPP from 100 to 125 and 150 mmHg were significantly augmented. AA diameters decreased by 14.8±1.5% and 24.0±3.4%, n=6, p<0.005 relative to responses during vehicle superfusion. We conclude that an augmented renal HO system in Ang II‐dependent hypertension contributes to attenuated renal autoregulatory capability. Supported by NIH grant HL26371, COBRE grant P20 RR 017659 and NKF postdoctoral fellowship
Localization and function of connexin 45 in the renal cortical vasculature
Authors: Hanner Fiona Pamela , Toma Ildiko , Sipos Arnold , Vargas Sarah , Peti‐Peterdi Janos
Published: June 2021

Connexins (Cx) play an important role in cell to cell communication via intercellular gap junction channels or transiently open pores (hemichannels). We studied Cx45 in the mouse kidney using conditional Cx45‐deficient (Cx45fl/fl:Nestin‐Cre) mice expressing EGFP in place of Cx45 in cells that during development expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriolar vascular smooth muscle (VSMC) and renin producing JG cells and in the extra and intraglomerular mesangium indicating that Cx45 is normally expressed in the juxtaglomerular apparatus (JGA). Immunoblotting detected a 1.5‐fold increase in renal cortical renin content in Cx45−/− mice. Wild type and Cx45−/− VSMCs were cultured from dissected afferent arteriole explants. Cells were loaded with Fluo‐4/Fura red and ratiometric [Ca2+]i imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single VSMC caused a 5‐fold increase in [Ca2+]i which propagated from cell to cell throughout the monolayer (12.6±1.1 μm/s) over distances >400 μm. In VSMCs from Cx45−/− mice Ca2+ wave propagation was significantly inhibited with velocity of 7±1 μm/s and peak [Ca2+]i =56.21±15 (n=6). Addition of either the purinergic receptor antagonist suramin or the Cx blocker 18‐alpha GA reduced Ca2+ wave propagations. Cx45 plays an important role in renal vascular control, renin release and JGA function.
Increasing participation of undergraduates from rural and tribal colleges in research
Authors: Cisek Karen , Boese Sarah , O'shea Lorraine , Bennett T.M. Bull , Pyle Sally , Van Doze
Published: June 2021

The undergraduate years are a critical time to engage students in research, but opportunities are sparse for rural and tribal students who are an underrepresented demographic in research labs. To reach this population, we have given special consideration to recruitment, lab experience, and retention. This has included partnering with successful Native American scientists and faculty at rural and tribal colleges, campus visits, and addressing the unique needs of this demographic. Utilizing a peer mentoring approach, we have trained 30 rural and tribal students in our lab and through a Research Experience for Undergraduates program. Peer mentoring in a research laboratory creates an environment which encourages students to continue in research. New students are paired with experienced undergraduates and gradually introduced to techniques and concepts. Undergraduates are supported in their strengths within the lab and encouraged to help spread their expertise to other students; advice and encouragement are plentiful from peers and faculty. This makes for a positive lab experience, promoting interest in research and science, and opening new career avenues. This approach has been effective, resulting in a significant number progressing to graduate or medical school, or secondary science education. Supported in part by ND EPSCoR, NSF CAREER 0347259, NSF REU Site 0639227, and the American Physiological Society.
Teaching Nematode Anatomy Online: WormAtlas and Slidable Worm
Authors: Hall David H , Crocker Chris , Norris Carolyn R , Herndon Laura A , Lints Robyn , Altun Zeynep F
Published: June 2021

We have constructed a website (www.wormatlas.org) which describes all cells and tissues of the hermaphrodite and male Caenorhabditis elegans for teaching purposes. New data will soon cover tissue development in the embryo. Imaging modalities used in the atlas include LM/DIC, confocal imaging, SEM, serial section electron microscopy (TEM), and illustrations depicting basic concepts and relationships for nematode development and physiology. Among various sections, the Handbook is designed as a text‐based resource with annotated images, which are color‐coded for individual tissues. The Slidable Worm uses Zoomify (open source) to view consecutive TEM sections of the worm at incremental magnifications, with or without color‐coded annotations. A Glossary defines all anatomical terms, with links to text and images. The site also offers an anatomical methods section, “neuron pages” describing individual cells of the nervous system, and HTML‐converted canonical anatomy texts. A printed version of the atlas is available as a portable resource (C. elegans Atlas, Hall and Altun, 2008, CSHL Press). Related websites that can be linked via the atlas offer catalogues of archival TEM images (www.wormimage.org) or GFP images (www.gfpworm.org), and a discussion forum for the worm community. Many C. elegans labs have supplied key data for this work, which is supported by NIH RR 12596.
Real‐time interactive fusion of physical/electronic auscultation simulation and 3D CT/MRI‐based anatomical modeling applied to training in lung sounds detection, discrimination and classification
Authors: Canby Craig , Hisley Kenneth , Kolbinger Gregory , Stanton Charles , Vardaxis Vassilios , Donald Matz , Khan Makdoom
Published: June 2021

Objective: To test the instructional effectiveness of simultaneous coupling of the spatial detection, discrimination and classification of simulated lung sounds to real‐time viewpoint tracking of displayed digital 3D anatomical models over the current method of auscultation alone. Methods: An integration of a computer‐based clinical training system comprising two complementary functions: 3D anatomical visualizations, and physiological diagnostic sounds simulation using a physical manikin. These functions are linked in real‐time by the student stethoscope's 3D position/orientation in space on the surface of the manikin. Thus, during any given simulation drill using as input a pre‐defined diagnostic lung sound, a student will sample signs from the manikin and simultaneously view the case's prescribed condition or pathology volumetrically reconstructed and visualized from de‐identified patient MRI/CT image sets carefully selected to represent the given abnormality. Expected results: Active student tracking, perception of diagnostic sounds and visualizations of the corresponding anatomical structures will significantly increase student learning over methods using auscultation without visualization.

This research is supported by the Department of Anatomy and the Dean's Office, Des Moines University.
Perceived Value of Computer‐Aided learning in a Gross Anatomy Course
Authors: Goldman Evan Michael
Published: June 2021

Results: Frequency of technology use by students was ranked using Likert scales as follows: (1) Never, (2) Rarely, (3) Sometimes, (4) Frequently, (5) Always. In response to questions regarding student use of CDs, DVDs, or software for studying for lecture or lab materials (four different questions) the averaged response was 1.70. Use of Internet resources for studying (lecture or lab) or online quizzes: averaged response from three questions, 2.39. Use of technology to help with dissections (one question), 1.26. Use of computers to access instructor's powerpoint lectures (one question), 3.82. In response to a second survey of “how useful” would these technologies be to their studying (Likert scale ranging 1‐4 with 1=not at all useful and 4=very useful), the averaged response (three questions) was 3.62. In a third survey, students were asked “how often” they would use technologies if they were available to them (Likert scale: (1) Never, (2) Rarely, (3) Sometimes, (4) Frequently, (5) Always). Use of a “searchable” anatomy atlas, radiographic atlas, or dissector available during lab/dissection sessions: averaged response of three questions, 3.99. Use of a “searchable” anatomy atlas, radiographic atlas, or dissector available outside of lab/dissection sessions: averaged response of three questions, 4.04.
The Role of CDK Inhibitor p21 in Anti‐proliferative Effects of 10‐Formyltetrahydrofolate Dehydrogenase
Authors: Hoeferlin Lauren Alexis , Oleinik Natalia V , Krupenko Sergey A
Published: June 2021

The folate enzyme 10‐formyltetrahydrofolate dehydrogenase (FDH) is reminiscent of a tumor suppressor because of its ability to inhibit proliferation of cancer cells and its strong down‐regulation in a variety of tumors. Furthermore, we have recently demonstrated that FDH expression in A549 lung cancer cells activates p53‐dependent apoptosis. Simultaneously, these cells displayed G1 arrest and accumulated p53 downstream target p21, an inhibitor of cyclin‐dependent kinases. To determine the role of p21 in FDH‐induced cell cycle arrest and apoptosis we evaluated effects of FDH in p21 deficient A549 cells. In these cells, p21 was silenced by targeting with short interfering RNA (siRNA). Overall, we have demonstrated that FDH expressing A549 cells, deficient in p21, exhibit accelerated apoptosis, apparently due to the elimination of a delay caused by cell cycle arrest. Indeed, we observed that effects of FDH on the cell cycle were abrogated by p21 silencing. It is not clear at present whether the role of p21 is specific towards FDH‐induced effects or it is a part of a more general response to alterations of folate metabolism. This question will be addressed in our ongoing experiments, in which we are testing effects of anti‐folates on p21 proficient and p21 deficient cells.
A transcription factor that senses fatty acid structure
Authors: Zhang Yong‐Mei , Zhu Kun , Frank Matthew W , Rock Charles O
Published: June 2021

Bacteria regulate their membrane biophysical properties by the coordinated synthesis of saturated and unsaturated fatty acids. In Pseudomonas aeruginosa, unsaturated fatty acids are produced by the de novo fatty acid biosynthetic pathway anaerobically, or by oxidative desaturation of the existing fatty acids catalyzed by Δ9 desaturases DesA and DesB. DesA introduces the double bond into phospholipid acyl chains, whilst DesB desaturates acyl‐CoAs. A transcriptional repressor DesT regulates the expression of desCB operon. The expression level of desCB is strongly and selectively up‐regulated in a ΔdesT deletion strain. Both electrophoresis mobility shift assay and DNase I footprinting analysis demonstrated the existence of two DesT binding sites in the desT‐desCB promoter region, P1 and P2. The binding of purified DesT to P2 was enhanced by unsaturated acyl‐CoAs, whereas saturated acyl‐CoAs prevented DesT interaction with P2. The biological importance of this interaction was verified by the up‐regulation of desCB in cells grown in the presence of stearate and their repression when oleate was present. This unique ligand selectivity allows DesT to sense the physical properties of the cellular acyl‐CoA pool and modulate the expression of the acyl‐CoA Δ9‐desaturase system accordingly. (Supported by GM34496 and ALSAC)
Transition metal induction of the S. cerevisiae OLE1 gene occurs through multiple promoter elements.
Authors: Mintz Jason D , Oh Chan‐Seok , Martin Charles
Published: June 2021

The S. cerevisiae Δ‐9 fatty acid desaturase, encoded by the OLE1 gene, converts saturated fatty acyl CoA to monounsaturated species. Hypoxia and transition metals, such as cobalt, induce OLE1 expression. Expression of OLE1 is dependent on two ER membrane proteins, Spt23p and Mga2p, that appear to serve as co‐transcription activators of OLE1, however, hypoxic and cobalt induction of OLE1 appear to be controlled only through Mga2p. The OLE1 promoter contains two regions essential for transcription activation. The FAR region activates transcription under normoxic conditions whereas the downstream LORE element is essential for the hypoxic stimulation of OLE1 expression under Mga2p activation. This study examines the role of cobalt on the Mga2p induction of OLE1 through the FAR and LORE promoter elements. In (spt23;MGA2) cells, cobalt causes a 25‐fold induction of the full promoter. Mutations in either the LORE or FAR region produce low, but cobalt‐inducible levels of reporter activity indicating that the two elements act in response to cobalt in a differential manner. Cobalt has been proposed to be linked through the hypoxic induction of OLE1 via its inhibition of a mitochondrial cytochrome dependent signal transduction pathway. Analysis of respiratory deficient mutants shows a strong cobalt activation of OLE1, however, suggesting that there may be more than one pathway for induction of the gene.
Desnutrin overexpression in adipocytes decreases triaclyglycerol content
Authors: Duncan Robin Elaine , Ahmadian Maryam , Sul Hei Sook
Published: June 2021

We recently identified desnutrin as a triacylglycerol (TAG) lipase that is mainly expressed in adipocytes. Desnutrin is induced by fasting when circulating levels of glucagon and glucocorticoids are elevated and lipolysis in adipocytes is high. While treatment with dibutyryl cAMP had no effect on desnutrin expression, dexamethasone caused a dose‐ and concentration‐dependent upregulation of desnutrin mRNA. Thus far, desnutrin has been shown to hydrolyze TAG in vitro, and to increase the release of free fatty acids and glycerol when overexpressed in cultured cells. However, the effect of desnutrin on TAG storage in adipocytes has not been investigated. We infected 3T3‐L1 ΔCAR adipocytes with adenoviral desnutrin‐GFP. We found that desnutrin‐GFP localized strongly to both large and small lipid droplets, and was also observed in the cytoplasm. In cells labeled with 14C‐palmitate, desnutrin significantly increased the rate of TAG hydrolysis. Desnutrin overexpression also significantly decreased TAG content under basal conditions as well as in cells stimulated with dibutyryl cAMP. Truncation of desnutrin resulted in impaired localization, as well as a loss of activity in differentiated 3T3‐L1 cells. Our findings indicate a mechanism to explain desnutrin‐associated neutral lipid storage disorder with myopathy, and may provide a target for the development of therapeutic treatments to counter this disease.
Gαo proteins regulate the α2A adrenergic receptor‐mediated inhibitory effects of epinephrine in the mouse hippocampal CA3 region
Authors: Nelson Brian W , Xu Ke , Goldenstein Brianna , Pribula Jacquline , Luger Elizabeth , Wald Jenna , O'Shea Lorraine , Charbeneau Raelene , Huang Xinyan , Richard Neubig , Doze Van
Published: June 2021

Activation of α2A adrenergic receptors by epinephrine (EPI) attenuates seizure activity in the rat hippocampal CA3 region. Questions concerning the specific mechanism of this action are whether the α2A receptors are located pre‐ or post‐synaptically, and what subtype of G protein is coupled to this receptor. Evidence suggests that pre‐synaptic terminals of the recurrent axon collateral on pyramidal cells are involved; we hypothesize that the α2A receptors are located pre‐synaptically. We used transgenic mice with knock‐ins of the inhibitory regulators of G‐protein signaling (RGS)‐insensitive G183S Gnai2 allele for either the pre‐synaptic Gαo or the post‐synaptic Gαi2 protein subtype. EPI's anti‐epileptic effects were assessed with electrophysiological recordings in brain slices from control mice, modified Gαo mice, and modified Gαi2 mice.

The EC50 of EPI was not significantly different between age‐matched C57BL/6J control and modified Gαi2 mice. In contrast, EPI was significantly more potent (>7‐fold) in the Gαo mice than the littermate controls. These results suggest that the pre‐synaptic Gαo protein and not the post‐synaptic Gαi2 protein mediates EPI's inhibition of hippocampal CA3 epileptiform activity. These findings could lead to new strategies for treating epilepsy. Supported by ND EPSCoR, NSF CAREER 0347259, NSF REU Site 0639227, NIH 5RO1GM039561, NIH P20 RR016741 from the INBRE Program, APS.
System xc‐ is Localized to Early Endosomes which Facilitates Regulated Membrane Trafficking
Authors: Goltz Abby , Wixson Matthew , Chase Leah A.
Published: June 2021

The membrane transporter, System xc−, catalyzes the exchange of cystine and glutamate across the membrane of many cell types. As such, this transporter plays significant roles in controlling intracellular cysteine levels and ultimately, the synthesis of glutathione, and regulating extracellular levels of glutamate, the primary excitatory neurotransmitter in the brain. Previous studies in our lab have demonstrated that hydrogen peroxide (whether added exogenously or produced endogenously) regulates the trafficking of System xc− to the plasma and that this process is responsible for maintenance of glutathione levels under conditions of oxidative stress. The objective of this study was to determine the mechanism by which peroxide regulates trafficking of System xc−. Using immunocytochemistry, we have demonstrated that System xc− is localized to early endosomes in naïve cells. Upon exposure to peroxide, however the endosomes appear to disperse, resulting an increase in System xc− expression on the membrane. We are currently using cell surface biotinylation assays to determine whether peroxide exposure increases the rate of delivery of System xc− to the membrane or whether it decreases the rate of removal (endocytosis) of System xc− from the membrane. This research was supported by the Campbell Foundation, the NSF REU and MRI Programs, and the Biology and Chemistry Departments at Hope College.
Phosphorylation and the Transcription Factor YY1
Authors: Rizkallah raed Mtanis , Hurt Myra M
Published: June 2021

Yin‐Yang 1 (YY1) is a multifunctional transcription factor that is involved in the regulation of a large array of genes controlling cellular proliferation, differentiation and even apoptosis. Although our knowledge of the events regulated by YY1 has grown significantly over the past decade, we still know little of what regulates YY1 and its functions. YY1 has been previously described as a phosphoprotein. However, only indirect evidence for this has been provided, and the role of phosphorylation in regulating YY1's functions has not been elucidated. Here we provide direct evidence for YY1's phosphorylation by in vivo labeling studies. Additionally we have studied the effect of phosphorylation on purified YY1's affinity to several of its DNA binding sites. Since YY1 is involved in the regulation of replication‐dependent histone genes at the G1/S transition of the cell cycle, we examined YY1 as a possible substrate for cell cycle kinases. We found that YY1 is a substrate for CyclinE/Cdk2 in an in vitro kinase assay. Through site directed mutagenesis, we have identified the phosphorylated amino acid. Phosphorylation of this site was independently confirmed in vivo by Mass Spec analysis of the purified protein. The function of phosphorylation of YY1 at this site is the subject of current research in our lab.

This research was supported by a grant from Order of the Eastern Star and FSU College of Medicine.
Upregulation of the alpha7 nAChR in human macrophages is induced by chronic treatment with HIV‐1 envelope protein, gp120
Authors: Lázaro María Isabel , Santiago‐Pérez Laura Iris , Ballester Leo , Román Laura Rodríguez , Azoulay Hilla , Lasalde‐Dominicci José A , González Carlos I
Published: June 2021

Human immunodeficiency virus type‐1 (HIV‐1) infection is the commonest cause of dementia in adults less than 40 years. The cellular and molecular alterations that result in synaptic dysfunction, neuronal degeneration and cell death due to HIV‐associated dementia (HAD) by HIV‐1 infection appears to be caused by the infection of macrophages, microglia or by stem cells. We examined the effects of gp120 on the upregulation of the alpha7 nAChR expressed in human macrophages. Compared to controls, chronic treatment with gp120 showed a remarkable increase in the expression of the α7 nAChR with an increase in the concentration of gp120. In addition, macrophages chronically exposed to gp120 showed an increase the macroscopic whole‐cell currents compared to controls. After chronic exposure to gp120, macrophages also presented an increase in calcium uptake compared to non‐treated macrophages. Interestingly, experiments in several cell lines showed a similar up‐regulation of the α7 AChRs, suggesting that the up‐regulation could be occurring through a direct mechanism. We are currently conducting polysome analysis in continuous sucrose gradients to determine whether treatment with gp120 enhances the α7 nAChR mRNA association with the translational machinery. This notable up‐regulation of the α7 nAChR induced by the gp120 may be critical to the functional state of macrophages. Our data suggests that the α7 nAChR could be a key player in the HIV pathogenesis.

This research work was supported by NIH GM008224, 2R01GM56371–11, NCRR 1S0RR13705 and by NIH SNRP‐U54NS4301 to JAL‐D. Also supported by NIH U54 Award CA96297–03 and NIH S06‐GM008102–3052. M.I.L is supported by RISE Award 2R25GM61151.
UV and H2O2 induce AMPK activation in cultured skin keratinocytes
Authors: Cao Cong , Lu Shan , Kivlin Rebecca , Amaral Ashley , Kouttab Nicola , Wan Yinsheng , Chu Wenming
Published: June 2021

AMP‐activated protein kinase or AMPK is an evolutionarily conserved sensor of cellular energy status, activated by a variety of cellular stresses that deplete ATP as well as other stresses. However, the possible involvement of AMPK in UV‐ and H2O2 ‐induced skin aging is not fully studied. We show for the first time that UV and H2O2 induce AMPK activation in cultured human skin keratinocytes (HaCaT cells). UV and H2O2 also induce LKB1, the possible upstream signal of AMPK, in an EGFR dependent manner. Using Compound C, a specific inhibitor of AMPK and AMPK specific siRNA knockdown as well as AMPK activator AICAR, we found that AMPK serves as a positive regulator for p38 and p53 (ser 15) activation induced by UV radiation or H2O2 treatment. We also observed that AMPK activation pathway serves a negative feedback signal pathway against UV‐induced mTOR activation in a TSC2 dependent manner. Inhibiting mTOR and activating p53 and p38 may mediate AMPK's pro‐apoptotic effect in UV or H2O2 treated cells. Furthermore, activation AMPK also phosphorylates acetyl‐CoA carboxylase or ACC, the pivotal enzyme of fatty acid synthesis, and PFK2, the key protein of glycolysis in UV‐radiated cells which may explain reduced adipogenesis in UV or H2O2 treated 3T3‐L1 adipocyte and skin keratinocytes. Collectively, our results demonstrate that UV and H2O2 induce EGFR/LKB1/AMPK signal pathway activation in cultured skin keratinocytes, which plays important role on UV or H2O2‐induced signal transduction and skin aging.
High affinity interactions promote T regulatory positive selection in the thymus
Authors: Haymaker Cara L. , Divekar Rohit , Cascio Jason , Yu Ping , Zaghouani Habib
Published: June 2021

Many autoimmune disorders have been linked to a lack in T regulatory cells. As such, an understanding of how these cells develop and mature is critical. While thymic selection of effector T cells has been described, this process remains largely undefined for Tregs. Since only the DM20 form of proteolipid protein (PLP) is expressed in the thymus during fetal and neonatal life, thymic selection against PLP139‐151, which is missing in DM20 is not operative during those periods. PLP139‐151 (designated PLP1) as well as PLP1 derived altered peptides with degenerate affinity to PLP1‐specific TCR were expressed on immunoglobulin (Ig) and the resulting chimeras were used to cross the maternal placenta, deliver the peptides from mother to fetus, and restore peptide selection during the fetal and neonatal period. This model was then used to determine whether Tregs are subject to thymic selection and to gauge the affinity required for such selection. The findings indicate that high affinity interactions promote the positive selection of Tregs. Indeed, Ig chimeras harboring high avidity peptides facilitated the positive selection of Tregs resulting in a higher number of Tregs progressing past the double positive stage in the thymus. However, chimeras incorporating low affinity peptide did not drive positive selection of Tregs in the thymus. Research supported by NIH grant NS37406.
Altered Adenosine to Inosine editing in lupus T cell transcriptome
Authors: Dama Laxminarayana
Published: June 2021

Adenosine to Inosine (A to I) editing induced by adenosine deaminases that act on RNA (ADARs) lead to the specific alteration of the primary sequence of an RNA molecule and its expression. Phosphodiesterase 8 (PDE8), Ubiquitin Carboxyl‐Terminal Hydrolase (UCTH) and Interferon alpha receptor 2 (IFNAR2) play an important role in regulating many cellular functions and their gene transcripts contain known editing sites. Therefore, in the present studies the transcripts of these genes were analyzed for ADAR mediated editing in normal and lupus patients. The principal finding of this study is the altered editing at known A to I editing sites of PDE, UCTH and IFNAR2 gene transcripts of lupus T cells. The second important observation is the occurrence of novel A to I editing sites and hot spots in these gene transcripts of the lupus patients. A third fundamental finding is the compensatory alteration of A to I editing frequencies at hot spots. Based on these results, it is surmised that, such alterations and compensations at selective editing targets in gene transcripts may be due to qualitative and/or quantitative changes in ADAR enzymes. Therefore, transcript editing is a novel unrecognized epigenetic mechanism that contributes to the abnormal expression of genes and, ultimately, impaired immunity in lupus pathogenesis.

This work was supported by National Institutes of Health Grant AR48628 (to D.L).
Delineating dynamics of mucosal dendritic cell networks via intravital microscopy
Authors: Velazquez Peter , Dustin Michael L
Published: June 2021

The gut associated lymphoid tissue maintains homeostasis with normal enteric flora while remaining primed to mount a protective response against pathogens. Recent studies have utilized genetic and intravital microscopic approaches to describe the role of dendritic cell (DC) subsets in protection from infectious microbes. In this study, we apply intravital microscopy to delineate mucosal DC dynamics. Using animals with a targeted replacement of a single allele of cx3cr1 with egfp, we visualize a single network of DCs spanning the superficial villus into deep lamina propria (LP). In small intestine, villus LP DCs overlap to form a meshwork of cells probing the microenvironment, similar to DCs in Peyer's Patch (PP) T cell zone. In deep LP, DCs form a network of circular structures, likely due to the presence of glands, with each unit comprised of 6 or fewer DCs. In large intestine, the network consists exclusively of circular structures. All DCs visualized are closely associated with vasculature. Interestingly, DC trafficking is not seen during homeostasis. Preliminary studies of T cell migration reveal that effector and regulatory T cells traffic along the same micro‐anatomic locale as the DC network. Together, this study highlights the extent and dynamic nature of the mucosal DC network and suggests the presence of a mucosal “highway” which facilitates interaction between DCs, effector T cells and regulatory T cells.
M cell development occurs preferentially in distal small intestinal Peyer's patches in mice lacking B cells (μMT), CCR6, or TRANCE/RANKL
Authors: Freel Kathryn A , Taylor Rebekah T , Butler Betsy R , Kumar Nachiket , Williams Ifor R
Published: June 2021

Peyer's patches (PP) are inductive sites for generation of mucosal IgA antibody responses in the small intestine (SI). M cells are specialized epithelial cells present in the follicle‐associated epithelium (FAE) of PP that acquire small particulate antigens and convey them to immune cells in the subepithelial dome. The exact molecular signals driving differentiation of FAE enterocytes into M cells have not yet been defined. In wild type (WT) mice, the extent of M cell development is equal in PP throughout the SI. B cell deficient mice (μMT) were previously shown to have smaller PP with fewer follicles and a reduced density of M cells. Whole mount immunofluorescence staining of PP from μMT mice with Ulex europaeus lectin (UEA‐1) labeled with rhodamine reveals a consistent proximal to distal gradient in the extent of M cell development with PP follicles from the proximal third of the SI containing 6.0% of the M cells in WT PP and follicles from the distal third containing 79.0% of the WT level. A proximal to distal gradient was also observed in 2 other mutant mice (CCR6 and TRANCE/RANKL knockout mice) with impaired M cell development. In TRANCE deficient mice no M cells are observed in PP from the proximal third of the SI and the number of M cells in the middle and distal third rises to just 1.9% and 12.3% of WT levels. The existence of a proximal to distal gradient in M cell development in PP from these mutant mice demonstrates that M cell development is a complex process shaped by molecular cues that differ regionally along the SI. The greater reduction in M cells in the proximal PP of the models studied may reflect the later initiation of PP organogenesis and/or the increased density of the enteric microflora in the distal SI.

Supported by the NIH (DK‐64730, DK‐64399).
Estradiol induces NKG2D ligand expression in human endometrial epithelium
Authors: Basu Satarupa , Pioli Patricia , Eriksson Mikael , Conejo‐Garcia Jose , Wira Charles R. , Sentman Charles L.
Published: June 2021

The human endometrium undergoes cyclical changes regulated by sex hormones. Evidence suggests sex hormones regulate NK cell recruitment into the uterus, where NK cells are found in large numbers. NKG2D is a dominant activating receptor expressed on human NK cells, γδT cells and CD8 T cells, and NKG2D ligands are known to be sensors of cellular “stress”. In this study, we investigated whether sex hormones directly regulate NKG2D ligand expression. Real‐time PCR analysis showed that NKG2D ligands were expressed in the endometrium, and there was a trend for greater micA expression in the secretory phase endometrium. NKG2D ligands were detected primarily on epithelial cells, and greater MICA expression was observed in immunohistochemical analysis of tissues from patients in the secretory phase. Estradiol increased MICA expression on uterine epithelial cells, and this regulation was estrogen receptor dependent. Uterine NK cell cytotoxicity of these epithelial cells was NKG2D dependent. These data suggest that estrogens regulate uterine NK cell activity by altering NKG2D ligand expression in the human endometrium. These findings have implications for hormonal regulation of NKG2D recognition in other tissues and diseases. This study was supported by a grant from the National Institutes of Health (AI50178)
Oral rice‐based non‐toxic mutant cholera toxin induces enterotoxin neutralizing immunity
Authors: YUKI Yoshikazu , TOKUHARA Daisuke , NOCHI Tomonori , YASUDA Hiroshi , MEJIMA Mio , KUROKAWA Shiho , TAKAIWA Fumio , KIYONO Hiroshi
Published: June 2021

Oral administration of rice‐expressed cholera toxin B subunit (CTB) has been shown to induce enterotoxin neutralizing immunity. We examined here whether oral vaccination of transgenic rice seed expressing nontoxic double mutant cholera toxin (dmCT), which consisted of two amino acid substitutions of ADP‐ribosyltransferase active center (E112K) and carboxyl terminal KDEL (E112K/KDGL) in A subunit (CTA) would effectively induce enterotoxin‐neutralizing immunity. The CTA and CTB in dmCT were expressed in rice seed under the control of the endosperm‐specific expression promoter 1.4Kb glutelin GluB‐3 and 2.3Kb glutelin GluB‐1, respectively. Using densitometry analysis, we found that an average 3 microgram of dmCT was stored in a seed. Western blot analysis under nonreducing conditions revealed that CTB expression in rice formed a pentamer but it was not associated with A subunit. When mucosally fed, the rice‐expressed dmCT induced CTB specific serum IgG and mucosal IgA antibodies with neutralizing activity but not any CTA specific serum and mucosal antibodies. These findings showed that the rice‐expressed dmCT generated protective immunity against cholera toxin after oral vaccination.
Rapamycin impairs skin γδ T cell function resulting in autophagy
Authors: Jameson Julie , Mills Robyn , Podshivalova Katie
Published: June 2021

Epithelial tissues provide barrier functions vital to the survival of the organism. To maintain this protective role, there is a complex regulation of epithelial cell proliferation and survival. γδ TCR‐expressing lymphocytes reside within the epithelia where they regulate tissue homeostasis and repair by producing growth factors, cytokines, and chemokines. Chronic nonhealing wounds are a devastating consequence of defective tissue repair. Patients that receive the immunosuppressant Rapamycin for the prophylaxis of acute kidney rejection report an increased incidence of wound healing complications. We hypothesize that rapamycin not only targets allograft‐specific αβ T lymphocytes, but also suppresses skin γδ T cells resulting in defective tissue repair. Rapamycin inhibits the signaling molecule mTOR, a key player in growth factor and cytokine signal reception. We have established a murine model of rapamycin administration in which there is delayed wound repair. Treatment with rapamycin arrests skin γδ T cell proliferation in G1 phase and causes the T cells to undergo autophagy. In addition skin γδ T cells exhibit a defect in TNF‐α production at the wound site. We propose that rapamycin blocks the ability of skin γδ T cells to receive nutrient and growth factor signals resulting in autophagy. This limits the ability of skin γδ T cells to proliferate and negatively impacts their ability to mediate wound repair.

Support from Leukemia and Lymphoma Society and NIH DK073098.
IFN‐γ secretion at effector immunological synapses (IS) in vivo
Authors: Lowenstein Pedro R , Barrett Robert , Puntel Mariana , Yang Jieping , Sanderson Nicholas , Bondale Niyati , Kroeger Kurt M , Castro Maria G
Published: June 2021

In vitro, IFN‐γ secretion is focused towards the immunological synapse (IS), while secretion of TNF‐α is multi‐directional. We have previously demonstrated the polarization of IFN‐ γ within T cells establishing Kupfer‐type or non‐Kupfer type IS with virally infected astrocytes in vivo. We developed a novel adenoviral vector system containing an IFN‐ γreporter to identify IFNγ secretion at immunological synapse in vivo. Astrocytes were infected with this adenovirus, and 30 days later we induced a systemic immune response against Ads, and 14 days after the immunization animals were sacrificed and brains studied by immunohistochemistry and confocal microscopy. Brain sections were stained for TCR, LFA‐1, TK, and IFN‐ γ. Confocal analysis confirmed IFN‐γ polarization, and quantitative analysis was used to demonstrate the expression from the IFNγ ‐reporter adenovirus in astrocytes of immunized mice in contact with CTLs, when compared to non‐immunized mice. These data suggests that IFN‐γ is secreted across the immunological synapse in vivo towards the target virally infected cell.

This work was funded by grants from NINDS, NIH.