bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium Regulation of Group VIA “Calcium‐Independent” Phospholipase A2
Authors: Hsu Yuan‐Hao , Dennis Edward A.
Published: June 2021

The Group VIA‐2 calcium‐independent phospholipase A2 (GVIA‐2 iPLA2; iPLA2β) is composed of seven consecutive N‐terminal ankyrin repeats, a linker region, and a C‐terminal phospholipase catalytic domain. Unlike cytosolic GIVA PLA2 or secreted GV PLA2, there are no known calcium binding sites on iPLA2 (Burke, JE, etc. 2009 JLR). Consequently, the activity of iPLA2 does not require calcium. We previously reported that ATP activated GVIA‐2 iPLA2 activity (Ackermann, EJ. etc. 1994 JBC; Lio YC. etc. 1998 BBA). In new experiments, ATP activated GVIA‐2 iPLA2 with a Kd = 0.9 μM in the mixed‐micelle assay. Calcium inhibited GVIA‐2 iPLA2 in an ATP dependent manner, indicating calcium interfered with the ATP binding to iPLA2. We have constructed a structural model of iPLA2 and verified it by Hydrogen/Deuterium Exchange‐Mass Spectrometry (DXMS) (Hsu, YH, etc. 2009 JBC). DXMS showed ATP bound to the ankyrin repeats 3–5 (regions 207–226, 216–226, 238–240 and 277–299) and caused a decrease of the deuteration rates in H/D exchange in these neutrally to positively charged ankyrin repeats. ATP binding also caused conformational changes on the n‐terminal pseudo‐ankyrin repeats regions (regions 84–104, 87–104, 94–104, 115–125 and 116–125). We also found that calmodulin inhibited iPLA2 at an IC50 of 1.2 μM in the existence of 500 μM calcium and 500 μM ATP. Calmodulin and calcium both inhibited GVIA‐2 iPLA2, but ATP prevented the inhibition by both calmodulin and calcium. NIH GM 20501
A comparative study of the lipid composition of the brain of chicken and rat during myelination; A chromatographic‐densitometric analysis.
Authors: Helmy Fatma Mahmoud , Morris Aaron
Published: June 2021

The lipid profile of brain from 10 day and 18 day old chick embryo, one day old chick and adult chicken (10 weeks old), as well as full‐term fetus rat, 21 day old male rat, young adult male rat, and pregnant female rat brain; were analyzed by thin layer chromatography and densitometry.

10‐day old chick embryo brain revealed a low concentration of GDG, n‐CMH, h‐CMH, and CMH‐S. The concentration of these glycolipids increased gradually as the chick embryo advanced in development, indicating age relatedness, and reached the highest level in the brain of adult chicken. Rat brain did not begin to show the presence of these lipids until at least 21 days old, possibly indicating that the nervous system of the bird model develops faster than that of mammals. In addition, phosphatidyl ethanolamine plasmalogen (PE), was shown to be the only alkenyl phospholipid in all samples analyzed. Two molecular species have been identified (PE1 and PE2), and PE2 has been shown to correlate with the myelination process. Phosphatidyl choline (PC), and sphingomyelin (SM), were also present. SM concentration increased gradually during development, and reached its highest level in adult chicken brain, as in rat brain.

The correlation of myelination, brain development and the level of these glycolipids, indicated the important role of these glycolipids in both brain maturation and function.
Nestling Oral Microbial Colonization in Tree Swallows (Tachycineta bicolor)
Authors: Danhof Heather Ann , Thorpe Patrick A. , Lombardo Michael P.
Published: June 2021

Microbes may have a positive impact on the fitness of birds through improving host nutrition and/or host pathogen defense. The oral microbial community of nestling Tree Swallows was characterized by sampling of the pharyngeal region. Nestling Tree Swallows were sampled at Nesting Day (ND) 3 and ND 18. Nestlings typically leave the nest box on ND19. Both parents were also sampled. DNA was extracted from samples and a hypervariable region of the 16s rRNA gene was amplified. PCR products were run on a denaturing gradient gel (DGGE) which separates similar sized products based on their individual sequence variation. We predict that parents and offspring will be more similar to each other than unrelated swallows due to transfer of microbes between mates and between parents and offspring. We also predict that adult‐like microbial communities will develop over time and show increasing similarity to adults by ND18. Characterization of the patterns within and between families as well as identifying microbes represented by specific bands will provide a direction for determining the influence of specific microbes on avian fitness. Funding provided by National Science Foundation S‐STEMS program, McNair Scholars Program, and GVSU Department of Biology.
Characterization of Multicopper Oxidase Related Protein (MCORP) in Two Insect Species
Authors: PENG ZEYU , Gorman Maureen J , Arakane Yasuyuki , Kanost Michael R
Published: June 2021

Multicopper oxidases (MCOs) have diverse functions but are evolutionarily and structurally related. Ten histidines and one cysteine form two highly conserved copper binding centers, which are required for oxidase activity. We discovered a gene that we named multicopper oxidase related protein (MCORP), which looks somewhat like a MCO, but lacks the conserved cysteine and many of the histidines. We identified MCORP orthologs in many insect species, including Tribolium castaneum, Anopheles gambiae, Aedes aegypti, Culex pipiens, Bombyx mori, and Acyrthosiphon pisum. Phylogenetic analysis of MCORPs and MCOs indicated that MCORPs belong to a specific cluster. We cloned full‐length cDNAs of A. gambiae and T. castaneum MCORP and studied their expression patterns by RT‐PCR. Both genes are highly expressed in late developmental stages, particularly in pupae. In adults, AgMCORP is expressed predominantly in reproductive tissues. We purified recombinant AgMCORP and TcMCORP to make antibodies that we will use to determine the location of MCORP within an insect. RNAi mediated knock down of MCORP in T. castaneum resulted in a pupal lethal phenotype, which demonstrated that TcMCORP is essential to pupal development. To try to learn the function of MCORP, we will expand our RNAi studies and determine whether recombinant MCORPs have enzymatic activity.

This research was supported by a grant from NIH (R01 AI070864).
Phosphorylation of Yin Yang 1 mediates fentanyl‐induced decrease in miR‐190 expression
Authors: Zheng Hui , Chu Ji , Zeng Yan , Loh Horace H , Law Ping‐Yee
Published: June 2021

G protein‐coupled receptor exhibits agonist‐selective signaling. μ‐opioid receptor agonists (morphine and fentanyl) utilize G protein‐ and β‐arrestin‐dependent pathways respectively in ERK activation, which leads to the differential regulation of microRNAs. microRNA microarray has identified miR‐190 as one microRNA under agonist‐selective regulation. Fentanyl, but not morphine, decreases hippocampal miR‐190 level in a concentration‐ and time‐dependent manner. Fentanyl suppresses miR‐190 expression by decreasing the transcription of miR‐190's host gene, talin2. Fentanyl‐mediated regulation of the Talin2 promoter activity is located at the − 208~− 200 region on the promoter. Mutating this region attenuated fentanyl‐induced decrease in Talin2 promoter activity. The − 208~− 200 region contains a binding site for Yin Yang 1 (YY1). YY1 overexpression increased, while YY1 downregulation decreased, the transcription of talin2. Fentanyl‐induced ERK phosphorylation led to YY1 phosphorylation, which decreased YY1's ability to stimulate talin2 transcription or to increase miR‐190 expression, by impairing its association with the − 208~− 200 region as demonstrated with the EMSA and ChIP assays. This study, therefore, delineates the signaling pathway in which fentanyl (but not morphine) regulates miR‐190 expression. (This research is supported in parts by a NIDA grant DA007339).
The Nuclear Protein Chromodomain Helicase Binding Protein 8 is a Novel A‐kinase Anchoring Protein
Authors: O'Donnell Maureen E , Russell Mary , Lund Linda , Zhang Yinghua , Bloch Robert , Bond Meredith
Published: June 2021

A‐kinase anchoring proteins (AKAPs) anchor the regulatory type II (RII) subunit of protein kinase A (PKA) via an amphipathic a‐helix. Using phage display, we isolated peptides from a chromatin remodeling protein, chromodomain helicase binding protein 8 (Chd8). Our objective was to characterize Chd8 as an AKAP. We analyzed isolated Chd8 peptides and found a candidate site in the amino terminus. We evaluated RII binding with far Western blotting, in which blots of a peptide encompassing this domain were incubated with purified RII. A probe for RII highlighted a band of the same molecular weight as the Chd8 peptide. Mutation of an isoleucine (I464P) within the putative Chd8 RII binding site, and addition of Ht31, a peptide that disrupts PKA:AKAP binding, eliminated this band. RII coimmunoprecipitated with the amino terminus of Chd8, but not the I464P mutant. This demonstrates a novel AKAP domain in Chd8. Using qPCR of embryonic, post‐natal, and adult heart, we demonstrated Chd8 mRNA decreases in development, but is present in adult tissue. Immunoblots and immunocytochemistry identified nuclear Chd8 in cultured cardiac myocytes. We predict that altered PKA binding to Chd8 in response to beta‐adrenergic stimulation regulates interaction of Chd8 with its binding partners and alters activation of gene transcription. This work supported by NIH/NHLBI T32 HL072751, NIH/NIA F31AG032162‐01, NIH R01 AG16613 and RO1 HL75016.
Effect of TBT exposures on Protein kinase D activity of human Natural Killer cells
Authors: Rana Krupa , Whalen Margaret M.
Published: June 2021

Natural killer (NK) cells are lymphocytes that target and destroy (lyse) tumor cells, virally infected cells, and antibody coated cells. Due to their capacity to respond without a need for prior sensitization, they are at the forefront of the immune response to tumor and virally infected cells. Tributyltin (TBT) is an organotin that is found in many different products. It is found in antifouling paints and has been used as an antifungal agent in a wide variety of applications including silicon baking parchments and shower curtains. Due to its contamination of the environment it has also been found in human blood. Exposure of human NK cells to TBT decreases their lytic function and causes activation of mitogen activated protein kinase (MAPK). The aim of the current study is to examine the role of the serine threonine kinase, protein kinase D (PKD) in the TBT‐induced activation of NK cell MAPKs. PKD activation has been shown in other cells types to lead to the activation of MAPKs. In this study NK cells were exposed to 300, 200, 100, 50, and 25 nM TBT for 10 min and 1 h and cell lysates were prepared and analyzed by SDS‐PAGE followed by Western blot. The activation state of PKD was monitored using an antibody to phospho‐PKD. The results indicated that PKD was activated approximately two fold by 300‐100 nM TBT within 10 min. These results suggest that TBT‐induced activation of PKD may be in part responsible for the TBT‐induced activation of MAPK.

Supported by NIH grant S06 GM008092‐34.
Decreased mineralization and lack of cyclin D1 response to Parathyroid Hormone Related Peptide (PTHrP) in primary osteoblasts isolated from MKP‐1 knock out mice
Authors: Datta Nabanita , Chaker Bayan , Abou‐Samra Abdul
Published: June 2021

MAPK phosphatase 1 (MKP‐1), a member of the dual‐specificity phosphatases, inactivate MAPKs via dephosphorylation of ERK, p38 and JNK. Whether MKP‐1 plays a role in PTH and PTHrP anabolic action in bone is yet to be investigated. Our previous study demonstrated a decrease in ERK‐MAPK and cyclin D1 and an increase in MKP‐1 expression in differentiated osteoblasts following PTH/PTHrP induction both in vitro and in vivo. To investigate the effect of MKP‐1 deletion in PTH1R anabolic action, primary calvarial osteoblasts were isolated from neonatal or 10‐week old MKP‐1 knock out (KO) and wild type (WT) mice via sequential collagenase digestion. The cells were differentiated with ascorbic acid (100μg/ml) and β‐glycerophosphate (5mM) and harvested between 7 to 21 days. Von Kossa staining at day 14 showed decreased mineralization in MKP‐1 KO osteoblasts compared to WT. In contrast to WT cells incubation of MKP‐1 KO osteoblasts with 100nM PTHrP had no effects on pERK or cyclin D1 expression. MicroCT analysis of mid‐diaphyses of tibiae and femora from 2 week old MKP‐1 KO mice showed decreased bone mineral density (BMD), bone mineral content (BMC) and tissue mineral content (TMC) compared to WT bone. Taken together, these results show that MKP‐1 may be important for PTH/PTHrP anabolic action in osteoblasts and play a role in the maintenance of bone mass.

(Supported by NIH R03 DE018245‐01 and R01DK53904)
Effects of high NaCl on cellular glutathione and glutathionylation of proteins in HeLa cells
Authors: Edwards Sarah K. , Burg Maurice B. , Dmitrieva Natalia I.
Published: June 2021

High NaCl causes oxidative stress, evidenced by increased cellular reactive oxygen species (ROS) and oxidation (carbonylation) of proteins. Reduced glutathione neutralizes ROS by becoming oxidized. Reversible glutathionylation of cysteines in proteins protects the proteins from irreversible oxidation and signals the redox state. We measured glutathione in HeLa cells and analyzed glutathionylation of proteins with anti‐glutathione antibody in non‐reducing Western blots. HeLa cells survive modest increases of NaCl (to 400–500 mosmol/kg), but die from apoptosis at 600–700 mosmol/kg. Acute increase to 400–500 mosmol/kg for 2 hours has little effect on the level of glutathione, but increase to 600–700 mosmol/kg decreases glutathione level at 1 hour, before the cells become apoptotic, and completely eliminates it by 2 hours, coincident with activation of apoptosis. Elevation of NaCl to 400–500 mosmol/kg, but not to 600–700 mosmol/kg, increases glutathionylation of a 40–45 kDa protein. Imminoprecipitation with anti‐glutathione antibody reveals several additional proteins whose glutathionylation increases at 400 mosmol/kg. We conclude that high NaCl‐induced apoptosis in HeLa cells is associated with a decreased level of glutathione, and we speculate that protein glutathionylation might contribute to signaling cellular adaptation to high NaCl. Supported by the Intramural Program of NHLBI.
A putative role for AHNAK1 in maintaining gastric epithelial permeability barrier properties
Authors: Alli Abdel A , Carter Gay M , Houyou Deborah M , Routen Eric S , Gower William R
Published: June 2021

The organization and integrity of the plasma membrane of gastric epithelial cells is crucial for providing a protective barrier for the stomach. AHNAK1 is a scaffolding protein that has been described as a protein marker of cells with barrier properties and is implicated in the regulation and organization of the cytoskeleton and the plasma membrane of epithelial cells. Previously, we reported AHNAK1 protein is expressed in different regions of the stomach of healthy human organ donors, and in cultured non‐transformed rat gastric mucosal (RGM1) polarized epithelial cells. We showed AHNAK1 protein is highly expressed in the cytoplasm and at the plasma membrane of RGM1 cells. Here, we investigated a possible function for AHNAK1 in mediating the selective paracellular permeability properties of gastric epithelial cells. An siRNA mediated knockdown of AHNAK1 protein in RGM1 cells by approximately 70% was determined by Western blot and densitometric analysis. Immunofluorescence studies revealed AHNAK1 protein colocalizes with the tight junction associated protein ZO2. In vitro cell permeability assays showed an increase in permeation of fluorescein isothiocyanate (FITC)‐dextran across RGM1 monolayers after transfecting cells with a smartpool of siRNAs targeting AHNAK1 compared to transfecting cells with nontargeting siRNAs or treating cells with EDTA. Taken together, our results suggest a role for AHNAK1 in maintaining the integrity of the permeability barrier properties of gastric mucosal cells. This research was supported by an American Heart Association Predoctoral Fellowship and a Veterans Affairs Merit grant.
Electrophysiological abnormalities in mice with genetic ablation of Rap1a GTPase
Authors: Jeyaraj Selvi C , Joshi Mandar S , Huang Hong , Unger Nicholas T , El‐Dahdah N. Paul , Wold Loren E , Strauch Arthur R , Elton Terry S , Quilliam Lawrence A , Bauer John A , Chotani Maqsood A
Published: June 2021

Rap1a, a member of the Ras‐like GTPase family, acts as a molecular switch coupling extracellular stimulation to intracellular signaling through cyclic AMP. The impact of Rap1 deficiency in heart function has yet to be defined. We tested the hypothesis that Rap1a null mice (Rap1A−/−) have abnormal ventricular excitation or contraction, using in vivo cardiovascular status measures by echocardiography, and ECGs (n=5/group) in comparison to wild‐type (WT) controls. At rest, no significant difference in heart rate, LV fractional shortening, or cardiac output, but slight reduction in systolic blood pressure were observed in the two groups. In contrast, significant increases in QT interval (41.4±4.1ms for WT and 57.6±2.5ms for Rap1A −/−; p<0.05), QRS duration (11.9±1.0ms for WT and 15.9±1.4ms for Rap1A −/−; p<0.05), and R‐R variability were observed in Rap1A−/− mice. Evaluation of the ERG1, long QT associated voltage‐activated potassium channel, with qRT‐PCR and Western blotting indicate a 2‐fold decrease in the mRNA and protein levels. Together, loss of Rap1A shows an impact on repolarizing mechanisms. Although the two Rap1 subtypes A and B are closely related, they are not functionally redundant, with Rap1A contributing to normal cardiac function. These findings suggest a novel genetic basis of prolonged QT interval and arrhythmia risks in vivo.
Rho signaling is involved in HIV‐1 Tat‐induced tight junction protein ZO‐1 translocation and alterations of claudin‐5 expression in brain endothelial cells
Authors: Zhong Yu , Hennig Bernhard , Toborek Michal
Published: June 2021

The blood‐brain barrier (BBB) is the critical structure preventing HIV trafficking into the brain. HIV Tat protein can contribute to the dysfunction of brain microvascular endothelial cells (BMEC) and facilitate HIV entry into the brain. Rho signaling plays an essential role in HIV‐1‐mediated disruption of the BBB integrity. Our data shows that Tat activated RhoA signaling pathway, leading to a decrease in claudin‐5 and ZO‐1 expression. ZO‐1 is known to link the trans‐membrane proteins occudin, claudins, and JAMs to many cytoplasmic proteins and to the actin cytoskeleton. The Rho cascades of small GTPases have gained considerable recognition as powerful regulators of actin cytoskeletal organization. However, the mechanism of Rho signaling, which regulates tight junction assembly, remains unclear. We hypothesize that ZO‐1 serves as a scaffold for signal transduction and organizing Rho signaling molecules with a transmembrane tight junction protein regulates Tat‐mediated tight junction dysfunction. Indeed, we found that Tat increased MLC and ZO‐1 translocation into nuclear as assessed by western blotting, and activate activator protein 1 (AP‐1) transcription factor JunD, resulting in a decrease in membrane tight junction protein claudin‐5 expression in BMEC. Moreover, inhibition of the Rho activation by the C3 exoenzyme blocks Tat‐induced ZO‐1 translocation and claudin‐5 disruption in BMEC. The present data indicates the importance of Rho signaling in regulation of ZO‐1activation modulating the integrity of the BBB in response to Tat exposure.
Requirement of c‐Src in Angiotensin II and Endothelin 1‐induced Activation of MAPKinases in Vascular Smooth Muscle Cells (VSMC)
Authors: Vardatsikos George , Kapakos Georgia , Srivastava Ashok K.
Published: June 2021

Endothelin‐1 (ET‐1) and Angiotensin II (Ang II), two important vasoactive peptides, have been implicated in the pathogenesis of vascular abnormalities such as hypertension and atherosclerosis, through the activation of growth promoting signal transduction pathways, which include mitogen‐activated protein kinases (MAPKs) and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (PKB). We have previously shown that c‐Src, a non‐receptor protein tyrosine kinase (NR‐PTK), is an upstream regulator of ET‐1 and Ang II‐induced activation of PKB in VSMC. However, the role of c‐Src in ET‐1‐induced MAPK signaling remains controversial in VSMC. Therefore, in the present studies, we have investigated the involvement of c‐Src in this process. Treatment of VSMC with ET‐1 and Ang II induced phosphorylation of ERK1/2, JNK and p38MAPK in A10 VSMC and in aortic VSMC isolated from 12 week old Wistar Kyoto rats (WK‐VSMC). PP2, an inhibitor of the Src family of NR‐PTK, decreased phosphorylation of ERK1/2, JNK and p38MAPK induced by both ET‐1 and Ang II in a dose‐dependent fashion. Furthermore, knockdown of c‐Src by siRNA also exhibited a similar response, and inhibited ET‐1 and Ang II‐induced ERK1/2, JNK and p38MAPK phosphorylation. In summary, these data suggest that both ET‐1 and Ang II signal the activation of MAPKs through a c‐Src dependent mechanism in VSMC. (Supported by grants from Canadian Institutes of Health Research).
Agonist‐induced Ca2+ mobilization is regulated by a complex involving Orai1, hTRPC3 and the type I inositol 1,4,5‐trisphosphate receptor
Authors: JARDIN ISAAC , WOODARD GEOFFREY E , LOPEZ JOSE J , SALIDO GINES M , ROSADO JUAN A
Published: June 2021

The nature of the Ca2+ channels involved in receptor‐operated Ca2+ entry (ROCE) and store‐operated Ca2+ entry (SOCE) are under investigation. Orai1 has been presented as a putative SOC channel and transient receptor potential (TRP) proteins as candidates to mediate SOCE and ROCE. Inositol 1,4,5‐trisphosphate receptors (IP3Rs) have been shown to interact with plasma membrane TRPC channels; however at present there is no evidence supporting the interaction between Orai and IP3Rs. The aim of the study was to investigate the interaction between Orai and IP3Rs and its role in agonist‐induced Ca2+ entry. Treatment with thapsigargin or cellular agonists results in association of Orai1 with the types I and II IP3Rs. In addition, hTRPC3, RACK1 and STIM1 interact with Orai1 upon stimulation with agonists. hTRPC3 expression silencing prevented the interaction of Orai1 with hTRPC3 and the association of Orai1 with the type I IP3R, but not with the type II IP3R, thus suggesting that hTRPC3 selectively mediates the interaction between Orai1 and the type I IP3R. hTRPC3 expression silencing attenuated ATP‐and CCh‐evoked Ca2+ release and entry. In conclusion, here we show that agonist stimulation results in the formation of a Ca2+ signaling complex including Orai1, TRPC3, RACK1 and type I IP3R, where hTRPC3 plays a key role, that is important for agonist‐induced both Ca2+ release and influx. Supported by MEC grant BFI2007‐60104.
S‐Adenosylmethionine (SAM) downregulates phosphodieterease 4B expression and attenuates endotoxin‐induced TNF expression in monocytes via cAMP/PKA pathway
Authors: Gobejishvili Leila , Barve Shirish , Kirpich Irina , Avila Diana , Joshi‐Barve Swati , McClain Craig
Published: June 2021

Elevated concentration of circulating TNFα occurring due to leaky gut and endotoxemia plays a significant role in the pathogenesis of several acute and chronic inflammatory disorders. Although SAM attenuates endotoxin stimulatable TNF production in vitro and in vivo, the underlying mechanisms are not fully understood. Our earlier work demonstrated that SAM decreases LPS‐inducible TNFα gene expression and attenuates organ injury. Further, our recent studies showed that PDE4B/cAMP pathway plays a critical role in the regulation of LPS‐inducible TNF expression. Accordingly, in the present study PDE4B expression was examined as the potential inflammatory mechanism targeted by SAM.

The results obtained demonstrate for the first time that exogenous SAM supplementation significantly lowered LPS‐inducible PDE4‐specific activity, increased cellular cAMP levels and consequently decreased LPS‐inducible TNFα expression. Further, the SAM/PDE4/cAMP effect on TNF expression was partially PKA dependent. Notably, these data identify PDE4 expression as a significant pathogenic component of endotoxin mediated inflammation which can be targeted by SAM for the potential therapeutic use in the treatment of diseases characterized by elevated TNFα expression.

This work was supported by NIH grants AA014371, AA015970, and Office of Dietary Supplements.
Ku and DNA‐PK dynamic conformations and assembly regulate DNA binding and the initial NHEJ complex
Authors: Hammel Michal
Published: June 2021

DNA double‐strand break (DSB) repair by non‐homologous end joining (NHEJ) is initiated by DSB detection by Ku70/80 (Ku) and DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) recruitment, which promotes pathway progression through poorly defined mechanisms. Here, Ku and DNA‐PKcs solution structures alone and in complex with DNA, defined by X‐ray scattering (SAXS), reveal major structural reorganizations that choreograph NHEJ initiation. The Ku80 C‐terminal region forms a flexible arm that extends from the DNA‐binding core to recruit and retain DNA‐PKcs at DSBs. Furthermore, Ku and DNA promoted assembly of a DNA‐PKcs dimer facilitates trans‐autophosphorylation at the DSB. The resulting site‐specific autophosphorylation induces a large conformational change that opens DNA‐PKcs and promotes its release from DNA ends. These results show how protein and DNA interactions initiate large Ku and DNA‐PKcs rearrangements to control DNA‐PK biological functions as a macromolecular machine orchestrating assembly and disassembly of the initial NHEJ complex on DNA.
Role of Dps1 and Dps2 in reactive oxygen species tolerance in Deinococcus radiodurans
Authors: Reon Brian Joseph , Grove Anne
Published: June 2021

Deinococcus radiodurans is an extremophilic bacterium, capable of enduring large doses of ionizing radiation, oxidative stress and prolonged periods of desiccation. D. radiodurans is also fairly unique in that it encodes two Dps (DNA protection during starvation) proteins, Dps1 and Dps2. We have characterized the Dps homologs and a comparison of their functionality is reported. Dps2 is able oxidize and sequester Fe2+ within its protein core. Also, Dps2 binds DNA, although with an apparent lower affinity than Dps1. Furthermore, Dps2 can protect DNA from reactive oxygen species, but is unable to protect DNA from endonucleases, while the converse is true for Dps1. Dps1 is solely capable of condensing genomic DNA in vivo, while notably Dps2 is non‐cytoplasmically localized, a characteristic yet seen of Dps proteins. Dps2 expression levels were shown to remain static throughout the cell cycle, in contrast to the massive upregulation seen for E. coli Dps. These findings suggest that Dps1 may serve a role in iron homeostasis and nucleoid organization, while Dps2 may protect D. radiodurans from exogenous ROS.
Saccharomyces cerevisiae mismatch repair complex MLH1‐PMS1 binds and rapidly diffuses along DNA in one dimension
Authors: Plys Aaron Jacob , Gorman Jason , Visnapuu Mari‐Liis , Greene Eric , Alani Eric
Published: June 2021

DNA mismatch repair is a conserved pathway that corrects replication errors introduced by DNA polymerase. Formation of an MSH‐MLH ternary complex at a mismatch results in the initiation of downstream repair events that act to excise the mismatch so that the template strand information is maintained. Previously we showed that the S. cerevisiae MSH2‐MSH6 complex can travel along the helical axis of undamaged DNA. This property is believed to allow the complex to efficiently locate lesions within the genome. Subsequent interactions with the MLH1‐PMS1 complex are poorly understood. To begin to investigate MSH‐MLH interactions, we have analyzed the behavior of MLH1‐PMS1 on DNA using bulk assays and total internal reflectance fluorescence microscopy (TIRFM) of single molecules. We observed a robust and stable DNA binding activity for MLH1‐PMS1 in the absence of MSH2‐MSH6. TIRFM experiments revealed populations of MLH1‐PMS1 that differ by the speed at which they travel along DNA. Some molecules move roughly the same speed as MSH2‐MSH6 molecules whereas on average others moved 10‐fold faster. Our data suggests that MLH1‐PMS1 can diffuse along DNA via hopping or stepping while topologically linked in a ring‐like configuration. We also show that MLH1‐PMS1 can freely bypass nucleosomes, providing the first direct demonstration that a passively diffusing protein can traverse a stationary nucleosome while traveling along a chromatin lattice. We are currently testing our hypotheses that the unstructured linker arms of MLH1 and PMS1 that connect their globular N and C terminal domains are important for rapid diffusion along DNA and traversing nucleosomes. This work was supported by the Howard Hughes Medical Institute, the NIH, and SUNY.
Interactions of yeast HMO2 with distorted DNA
Authors: Rahman Wedad , Ray Sreerupa , Grove Anne
Published: June 2021

High mobility group proteins are non‐histone nuclear proteins that play a role in DNA repair. The functional roles of HMO2, an HMGB homolog from yeast, were studied by substituting a valine in box A with an alanine. Sequence alignment predicts that this residue intercalates between DNA base pairs. A binding assay with linear DNA showed that the mutant protein has lower binding affinity compared to wild type HMO2, while both mutant and wild type proteins have similar affinity for supercoiled DNA. Both wild‐type and mutant protein were able to protect free DNA ends from exonuclease digestion, suggesting that the substitution does not affect the preferred binding to DNA ends. Assays involving DNA with 2 loops that were seven and nine base pairs apart showed that HMO2 preferred binding to the latter; the 9 bp distance between loops is apparently optimal for HMO2 binding. In a competition assay involving HMO2, supercoiled DNA was able to compete for DNA with 2 loops (9 bp apart) while linear DNA was not. Evidently, the valine in HMO2 box A is important to the protein's ability to bind to linear DNA while its substitution does not alter the preferred binding of HMO2 to DNA ends or to DNA with distortions.
Maturational alteration of differential expressions of GC:AT‐biased genotypes of Cordyceps sinensis fungi and Paecilomyces hepiali in Cordyceps sinensis
Authors: Zhu Jia‐Shi , Gao Ling , Yao Yisang , Zhou Yanjiao
Published: June 2021

We reported simultaneous detections of the rDNA of Paecilomyces hepiali (Ph) and Hirsutella sinensis (Hs) in Cordyceps sinensis (Cs), a precious Chinese herb, and the altered phenotypes of Cs mutants with Cs maturation. In this study we performed Southern blotting analysis and found dramatic maturational increases in the biomasses of Ph and Hs rDNA. Two moieties are shown on the Hs blot when the genomic Cs DNA was prepared with EcoRI. MassARRAY SNP genotyping revealed both GC‐ and AT‐biased mutants in Cs stroma. The AT‐biases did not express in premature Cs caterpillar, but highly predominately in premature stroma. The GC‐bias expresses oppositely in the premature Cs compartments. The differential expressions were altered non‐proportionally with Cs maturation. The expression of the AT‐biases was similar to that for Ph. When Cs stroma genomic DNA was used as the template, inversely orientated rDNA and EcoRI‐digestible PCR products within ITS regions were increased proportionally to the increases in stroma height, indicating Cs maturation‐related DNA mutations and reverse fusion of chromosomes. In conclusion, Cs maturation associates with augmented biomasses of both Ph and Cs mutant genes and altered differential expressions of Cs fungi with GC:AT‐biased genotypes, representing an element in Cs lifecycle important to the anamorph‐telemorph connection of this natural herb.
CpG‐DNA Protects Against UV‐Induced Apoptosis Via Enhancement of AKT and mTORC1 in Keratinocytes And Dendritic Cells
Authors: Lu Shan , Cao Cong , Kivlin Rebecca , Sarkisian Simon , Parisi Joseph , McCauley Michael , Nalbandian Ari , Brum Gabriella , Xu Aie , Chu Wen‐ming , Marshall John , Wan Yinsheng
Published: June 2021

CpG‐DNA and its related synthetic CpG oligodeoxynucleotides (CpG‐ODNs) play an important role in immune cell survival and activation. Existing data have shown that AKT activation is involved in this process. However, the potential role of CpG‐ODN in protection against UV‐induced skin damage has thus far not been tested. In this study, we found that CpG‐ODN protects against UV‐induced cell death and apoptosis in both skin keratinocytes (HaCaT cells) and cultured dendritic cells (XS106, or DCs). In mechanism research, we found that CpG‐ODN pre‐treatment enhances UV induced activation of AKT (ser473, thr308 phosphorylation) as well as downstream signal TORC1 (S6K and 4E‐BP1 phosphorylation) in both HaCaT cells and DCs. AKT deficiency (by using AKT1/2 double knockout Mouse Embryonic Fibroblasts as well as LY 294002, a pharmacological inhibitors of AKT) or mTORC1 inhibition (by using rapamycin, a pharmacological inhibitor of mTORC1) largely neutralize the protective effects of CpG‐ODN, indicating that AKT and downstream signaling mTORC1 activation are necessary for CpG‐ODN induced protective effects against UV induced cell death. Our findings suggest that CpG‐ODN may be utilized to prevent from UV‐induced skin aging and provide a novel mechanism of protective effects against UV radiation.
Discovering HDV/CPEB3‐like Ribozymes
Authors: Webb Chiu‐Ho Tsai , Riccitelli Nathan J , Ruminski Dana J , Lupták Andrej
Published: June 2021

HDV/CPEB3‐like self‐cleaving ribozymes are characterized by the conserved nested double‐pseudoknot structure. Secondary structure‐based searches were used to identify the RNAs capable of forming this intricate fold. Sequence searches based on identified ribozymes showed that plants, fungi, and a unicellular eukaryote also harbor the ribozymes. In vitro experiments confirmed the self‐cleavage activity in several eukaryotes, a bacterium, and an insect virus. Multiple copies, and in some cases multiple sequence families, of the ribozyme are found in most genomes, typically mapping to introns and intergenic regions. The in vitro self‐cleavage rates of ribozymes vary over more than three orders of magnitude with the shortest half‐lives being ~10 seconds at physiological conditions. Rapid amplification of cDNA ends (5′ RACE) and reverse transcription followed by quantitative PCR (RT‐qPCR) experiments show that mosquito Anopheles gambiae (Agam) ribozymes are expressed and self‐cleave in vivo. The Agam‐2 ribozymes are the largest identified HDV‐like ribozymes, containing long sequences in the J1/2 and P4 regions. Transacting ribozyme was constructed based on the Agam‐2‐1 sequence and efficient scission was observed. These results indicate that HDV‐like ribozymes are widely distributed in nature and may play a variety of the biological roles.
Knot your average RNA: The Chlorella virus pyrimidine dimer‐specific glycosylase intron
Authors: Larson Brian Craig , Burton Aaron S , Lehman Niles
Published: June 2021

In vitro transcriptions of the 98‐nucleotide intron found in the Chlorella virus pdg locus always produce at least one slower‐migrating species (as ~10–20% of the total transcription yield) in addition to the expected transcripts. Extensive characterization of these slower‐migrating products suggests that they are knotted RNA molecules, which we call knRNAs, previously observed only by the ligation of synthetic ribo‐oligonucleotides. An RNA with knot‐forming interactions would display physical properties unique to this topology. The observation that knotting cannot be induced upon renaturation of the full length intron argues that the co‐transcriptional folding pathway that allows knotting is inaccessible during post‐transcriptional folding. We propose that knotting is the result of co‐transcriptional mis‐folding that allows the 3′‐end of an intron to become threaded through an internal loop during formation of its pseudoknotted structure, resulting in an open knot. Because it occurs during a biologically relevant process, this phenomenon may occur in vivo as well.
In vitro selection of novel classes of GNRA tetraloop receptors
Authors: Calkins Erin Rebecca , Keleshian Vasken , Jaeger Luc
Published: June 2021

GNRA tetraloop receptors are prevalent self‐assembling structural motifs in natural RNAs. The most recurrent of these is the 11 nucleotide (11 nt) receptor, which has a high affinity and specificity for GAAA tetraloops. This modular receptor takes advantage of three submotifs; the A‐minor interaction, AA platform and UA handle. Currently, few natural tetraloop receptors are highly specific for tetraloops other than GAAA. An in vitro selection strategy was employed to investigate whether novel receptors for non‐GAAA tetraloops with similar binding modality could exist. Using partially randomized sequences that maintain the A‐minor interaction, a number of new receptors binding GGRA and GURA tetraloops were obtained. Based on their binding affinities and specificities, a structural network probing the sequence space of these novel classes of receptors was created. Analysis of the sequence variants demonstrated that the selected receptors, albeit different from the 11 nt receptor, still retain a similar structural organization. Funded by the National Institutes of Health (R01 GM079604‐01).
Analysis of ESTs for alternatively spliced 3′ and 5′ splice sites reveals optimal exon size in vertebrates
Authors: Shepard Peter Joseph , Hertel Klemens J
Published: June 2021

In vertebrates most exons are small surrounded by much larger introns. Due to this architecture, splice sites are initially recognized across the exon in what is known as exon definition. Here we examine the relative usage of 3,000 pairs of alternative 3′ splice sites and 2,000 pairs of alternative 5′ splice sites in the human genome. We find that within an optimal exon length window >50 <250 nts, there is a positive correlation between splice site strength and splice site usage. However, when one splice site is located inside of this exon length and the other is not, the splice site inside of the optimal exon length is preferred regardless of splice site strength. Our analysis reveals that splice sites within an optimal exon size are preferentially used when compared to splice sites outside of this optimal size. We conclude that a constraint of exon size is a fundamental parameter that dictates splice site usage. Because the spliceosome eventually forms across the intron, it is believed that intron definition is the predominant mode of splice site recognition for introns smaller than 250 nucleotides in length. However, in a subset of our alternative splice sites, in which one splice site is exon defined and the other is intron defined, the exon defined splice site is preferentially used, suggesting that exon recognition is the default mode of splice site recognition in vertebrates.

NIH
The influence of dietary lipids on mitochondrial hydrogen peroxide production in liver of calorie restricted mice
Authors: Chen Yana , Domer Keira , Hagopian Kevork , Ramsey Jon
Published: June 2021

Calorie restriction (CR) has been shown to delay the onset of age‐related diseases and prolong life spans in a variety of species. CR reduces mitochondrial reactive oxygen species production, attenuates oxidative damage and alters membrane composition towards saturation. This has led to a theory that changes in membrane saturation are central to the actions of CR. As a first step towards testing this theory, C57BL/6 mice were assigned to 3 dietary groups (control and two CR groups) and fed AIN‐93G diets at either 95% (control) or 60% (CR) of ad libitum. To manipulate membrane composition, the primary dietary fat for the CR groups were either soybean oil (also used in the control diet) or fish oil. The diets were fed for 1 month and then liver mitochondria were isolated for measures of mitochondrial lipid composition, proton leak, and H2O2 production. Results indicate that changes in mitochondrial lipids in the CR mice reflected the dietary lipid source. Body weight was lower (p<0.01) in the soy compared to fish CR mice. Mitochondrial H2O2 production was decreased (p<0.01) in the CR fish group compared to the control and CR soy groups regardless of the substrate used for respiration. The results of this study indicate that decreases in long chain unsaturated fatty acids are not necessary for reduced ROS production with CR. Diets containing fish oil may further decrease ROS production with CR. (Supported by NIH RO1 AG028125)
Cold treatment does not increase the rate of platelet clearance in 13‐lined ground squirrels.
Authors: Richters Karl E , Benrud Ryan , Hordyk Peter , Cooper Scott
Published: June 2021

Thirteen‐lined ground squirrels (Spermophilus tridecemlineatus)experience a dramatic decrease in body temperature and heart rate during hibernation. These extreme conditions would likely cause human platelets to clot due to low blood flow rate and pressure. We have found that ground squirrels drastically decrease their circulating platelet and leukocyte counts during hibernation, likely to reduce the risk of clotting. Previous research has shown that chilled human and mouse platelets are rapidly cleared from circulation. However, upon arousal from torpor, ground squirrel platelets are rapidly released into circulation from their storage site in the spleen and persist for several days until new platelet synthesis begins. Flourescently labeled platelets were incubated at 4°C or 37°C and reinjected into autologous ground squirrels. Clearance rates of both warm and chilled platelets were the same. Identification of protective factors that prevent rapid clearance of chilled platelets may ultimately lead to the ability to refrigerate human platelet concentrates. This work was supported by an NIH R15 grant.
Vitamin A and vitamin E levels in lung from subjects with chronic obstructive pulmonary disease with different degree of severity of emphysema
Authors: Schäffer Michael W , Roy Somdutta Sinha , Das Salil K
Published: June 2021

Vitamin A (VA) and its active metabolites play an essential role in the respiratory tract by influencing differentiation and the integrity of the epithelium. A study that attempted to relate VA status with the degree of lung airway obstruction, revealed a lower serum retinol concentration in patients with moderate to severe COPD. Further, treatment with retinol improved the FEV1 in subjects with COPD.

To test our hypothesis that VA signaling pathway is compromised in COPD, we analyzed human lung samples from COPD patients, differing in the degree of severity of emphysema, provided by the Lung Tissue Research Consortium for their VA and tocopherol (T)‐levels by HPLC. T levels were determined since it is known that α–T is an inhibitor of retinyl ester hydrolase activity. VA levels (retinol and retinyl ester) were found to decrease with severity of the disease from mild (168 ng/g, 160 ng/g), to moderate (136 ng/g, 99 ng/g), and to severe (127 ng/g, 71 ng/g). α‐T levels were less in moderate (12.8 μg/g) and severe (13.7 μg/g) groups than that in mild group (15.5 μg/g). Similarly δ‐T‐levels were less in moderate (229 ng/g) and severe (142 ng/g) groups than that in mild group (231 ng/g). However, it is interesting to note that γ‐T levels were higher in moderate (4.6 μg/g) and severe (2.2 μg/g) groups than that in mild group (1.3 μg/g). Results indicate an involvement of VA & Ts in COPD. (Supported by NIH grant 1RO3 HL095419‐01A1).
Impact of intracellular glycosylation on the pro‐survival role of insulin
Authors: Teo Chin‐Fen , El‐Karim Enas Gad , Wells Lance
Published: June 2021

O‐GlcNAc modification on the serine and threonine residues of intracellular proteins is an inducible and dynamic glycosylation catalyzed by a pair of cycling enzymes, O‐GlcNAc transferase (OGT) and β‐N‐acetylglucosaminidase (OGA). Previous studies have linked an elevation of global O‐GlcNAc level to the development of insulin resistance in cell culture and animal models via perturbing insulin‐stimulated glucose uptake. In addition to the glucose homeostasis, insulin resistance also affects the anti‐apoptotic action of insulin in the neuronal system leading to retinopathy in type 2 diabetic individuals. Here, we report that increased global O‐GlcNAc levels, via pharmacologically inhibiting OGA or genetically overexpressing OGT, antagonizes the pro‐survival action of insulin in cell culture models. These observations are correlated with the down‐regulation of signal transduction through the metabolic branch of the insulin pathway, resulting in a reduction in Akt activation. While many proteins involved in the insulin mediated Akt activation are O‐GlcNAc modified, we consistently observe an O‐GlcNAc‐dependent alteration in the phosphorylation on IRS proteins. We conclude that O‐GlcNAc modification on IRS proteins may serve as the major functional target for fine‐tuning this signaling event. This work is supported by NIDDK/NIH RO1 DK075069 (LW). CFT is an AHA pre‐doctoral fellow (0715377B).
Mitochondrial de novo thymidylate biosynthesis
Authors: Anderson Donald Dean , Quintero Cynthia Marcela , Stover Patrick J
Published: June 2021

The de novo thymidylate (dTMP) pathway in mammals consists of serine hydroxymethyltransferase (SHMT1 and SHMT2Î± ), thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR). Recent work in our lab has shown that the de novo dTMP pathway is localized to the nucleus in S and G2/M phases and that nuclear de novo dTMP biosynthesis occurs in purified mouse liver nuclei. We have observed that decreasing levels of SHMT1 in the nucleus results in a depletion of nuclear dTMP synthesis and increases in uracil misincorporation. Increased uracil misincorporation occurred although cytosolic SHMT1 levels were increased six‐fold. This suggests an important role for nuclear de novo dTMP biosynthesis in inhibiting uracil misincorporation. These data suggest that de novo dTMP synthesis for nuclear DNA replication may occur exclusively in the nucleus. In this study, the ability of mitochondria to conduct de novo dTMP synthesis for DNA synthesis in that compartment was investigated. We have now shown the localization of TYMS, DHFR, and SHMT2 in the mitochondria of HepG2 cells using confocal microscopy and IPs of purified HepG2 mitochondrial fractions. DHFR activity was also observed in mitochondrial lysates suggesting that de novo dTMP biosynthesis can also occur in mitochondria. Collectively, these studies demonstrate that cytoplasmic dTMP synthesis is not necessary to support DNA replication.
C. elegans homolog of tailless regulates male gonadal migration
Authors: Kato Mihoko , Sternberg Paul
Published: June 2021

Cell migrations are vital for the development of multicellular organisms and require precise spatial and temporal regulation. Interestingly, the migratory cell itself undergoes change during its migration, so temporal development is an important consideration in examining its dynamic cell biology. We are studying the male linker cell, an individual cell in C. elegans that directs the development of the male gonad by carrying out a complex, long‐range migration. During the mid‐third larval stage, we observed the downregulation of one of two C. elegans netrin receptors, unc‐5. We show that this downregulation is required for the linker cell to respond differentially to netrin, an extracellular matrix protein present along the ventral bodywall when turning. During the fourth larval stage, we observe changes in linker cell shape, the membrane localization of the Rac homology, MIG‐2, and the expression of a zinc metalloprotease, zmp‐1. In an RNAi screen of 508 transcription factors, we identified nhr‐67, the ortholog of the Drosophila tailless and vertebrate Tlx genes, to be required for normal linker cell migration. We show that nhr‐67/tailless/Tlx functions cell autonomously in the linker cell to execute each of the third and fourth larval stage changes described. We propose that a series of transcriptional regulators, like nhr‐67/tailless/Tlx, controls temporal subsets of the linker cell migration program.