bluuebunny/crossref_metadata_2025 huggingface dataset:
Phospholipase D and Phosphatidylinositol 3‐kinase are Activated by Oxidative Stress in Central Nervous System.
Authors: Salvador Gabriela Alejandra , Uranga Romina María , Mateos Melina Valeria , Giusto Norma María
Published: June 2021

The purpose of the present work was to study the involvement of phospholipase D (PLD) and phosphoinositide‐3‐kinase (PI3K) signaling in synaptic endings incubated under oxidative stress injury. For this purpose, synaptosomes purified from rat cerebral cortex were exposed to the oxidative insult (FeSO4, 50 μM) or vehicle and subsequently phosphatidylethanol (Peth) and phosphatinolsitol‐3‐phosphate generation were evaluated. Peth formation was increased after 5, 30, and 60 min of Fe2+‐exposure with respect to the controls, whereas PI3P levels were only increased after 5 min of Fe2+ exposure. PI3K activity was also measured by using anti‐p85 immunoprecipitates. Peth generation was not altered by the presence of the tyrosine kinase inhibitor, genistein, whereas PI3K activation was dependent on tyrosine phosphorylation. Synaptosomal viability was evaluated by using MTT reduction and LDH leakage measurements under all experimental conditions. These viability parameters were significantly affected in the presence of FeSO4, with respect to control conditions. Neither ethanol nor LY294002 were able to prevent the deleterious effect of free iron. Our results demonstrate that oxidative stress activates PLD and PI3K pathways in cerebral cortex synaptic endings.

Supported by Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional del Sur and Agencia Nacional de Promoción Científica y Tecnológica.
25‐Hydroxycholesterol 3‐sulfate regulates lipid metabolism via SREBP‐1 in human macrophages
Authors: Li Yunzhou , Ma Yongjie , Hylemon Phillip , Zhou Huiping , Pandak William M , Ren Shunlin
Published: June 2021

Macrophages are the most prominent cell type in atherosclerotic lesions. The earliest atherosclerotic lesions are characterized by the accumulation of foam cells in the arterial wall. The foam cells develop following the accumulation of lipids in macrophages. To reduce the accumulation or increase the elimination of lipids in macrophages is central to prevent the development of atherosclerotic foam cells. We have found that 25‐hydroxychosterol 3‐sulfate (25HC3S) plays an important role in lipid metabolism in hepatocytes. In the present study, we further examined the role of this oxysterol in lipid metabolism in macrophages. The results indicate that treatment of human leukemia cell line derived macrophages with 25HC3S decreased expression of sterol regulatory element binding protein and of its downstream targeting genes encoding key enzymes, fatty acid synthase (FAS), acetyl CoA carboxylase (ACC‐1), and HMG reductase, which are involved in fatty acid and cholesterol biosynthesis; Oil‐Red O staining and MTT cell proliferation assay showed that 25HC3S decreased intracellular lipid accumulation and increased the cell viability in the oxidized low density lipoprotein‐treated cells, which were opposite to the effect of 25‐hydroxycholesterol. These studies suggest that 25HC3S is a potent regulator of lipid metabolism in macrophages and may be useful for prevention and therapy of atherosclerosis.
Structural basis for the crypticity of B cell epitopes of the Goodpasture autoantigen: The role of novel collagen IV crosslinks
Authors: Vanacore Roberto Marcelo , Ham Amy Joan , Todd Parvin , Borza Dorin Bogdan , Hudson Billy G
Published: June 2021

The autoantigens of certain autoimmune diseases harbor B cell epitopes that are sequestered by the antigen's tertiary or quaternary structure. The exposure of such cryptic epitopes, prompted by pathophysiological conditions, may be an important feature in the etiology of autoimmune disease. Here, we investigated the structural basis for the crypticity of B cell epitopes of the autoantigen of Goodpasture disease, an autoimmune disorder that causes rapidly progressive glomerulonephritis and pulmonary hemorrhage. The native autoantigen is the α3α4α5 noncollagenous hexamer, a globular domain that connects two triple‐helical protomers within the collagen IV network of basement membranes. The complete crypticity of autoepitopes within the hexamer correlates with the presence of dimer subunits. Here, the chemical basis for dimer formation was determined. Mass spectrometry analyses demonstrate that the α3α5‐heterodimers are connected by s‐hydroxylysyl‐methionine crosslinks, whereas, the α4α4‐homodimers are connected by s‐lysyl‐methionine crosslinks. These crosslinks function as molecular fasteners that reinforce the hexamers and block the accessibility of autoantibodies rendering the epitopes completely cryptic. Perturbation of the assembly or cleavage of crosslinks may be a key factor in the etiology of Goodpasture disease
Roles of two GDP‐mannose transporters in cell wall stability in the filamentous fungus Aspergillus nidulans
Authors: Gordon Barbara , Fay Lauren , Martin Stuart , Nkashama Sonia , Patel Ravi , Hill Terry W. , Loprete Darlene M. , Jackson‐Hayes Loretta
Published: June 2021

In order to identify genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans. We have identified two distinct genes (GMT1, GMT2) in one such mutant (R205) that are homologous to the yeast VRG4 GDP‐mannose transporter genes. GDP‐mannose transporters carry nucleotide sugars from the cytosol across the Golgi apparatus membrane in various eukaryotic organisms including plants and a variety of fungi. We have observed punctate patterns of fluorescence of GMT1 and GMT2 GFP chimeras, indicative of Golgi localization in A. nidulans. Cloned GMT1 and GMT2 complement hypersensitivity to the chitin binding agent calcofluor white in strain R205, and ameliorate hyper‐branching and septation abnormalities R205 displays under normal growth conditions. GMT1 in R205 contains a genetic mutation that causes an A315P amino acid substitution, and the amino acid sequence of GMT2 in R205 is identical to wild type. However, both GMT1 and GMT2 appear to be essential. These findings suggest that GMT1 and GMT2 are involved in cell wall maintenance in A. nidulans.

Funded by Research Corporation and the Merck/AAAS Undergraduate Science Research Program
Structure and function of the Gβ5‐RGS7‐R7BP complex
Authors: Sandiford Simone L. , Wang Qiang , Slepak Vladlen Z.
Published: June 2021

Regulators of G protein signaling (RGS) proteins accelerate the deactivation of G proteins by acting as GTPase activating proteins (GAPs) towards Gα subunits. Along with a RGS domain, members of R7 subfamily of RGS proteins contain a Dishevelled, Egl‐10, Pleckstrin (DEP) and Gγ Like (GGL) domains. Through the GGL domain they form a stable complex with the Gβ5 subunit. The DEP domain binds to R7 binding protein (R7BP) which recruits the complex to the plasma membrane. Recent studies identified a novel interaction within this complex: between the DEP domain and the Gβ5 subunit. In this study, we utilized site directed mutagenesis, molecular modeling, GST pulldown assay, FRET analysis, fluorescence microscopy and the calcium mobilization assay to study the structural features and functional role of the Gβ5‐RGS7 complex. Based on these studies we conclude that disruption of the Gβ5‐DEP interaction increases the ability of the Gβ5‐RGS7‐R7BP complex to inactivate signaling from M3 muscarinic receptor. We also found that Gβ5‐RGS7 inhibits calcium mobilization in a receptor‐dependent manner and examined the mechanism of this inhibition. In addition to the studies at the molecular and cellular level, we investigated metabolism and behavior of mice with an altered expression of the Gβ5‐RGS7 complex. This research is supported by NIH grant GM 060019 (V.Z.S.).
Transmembrane domain 1 of human dopamine D1 and D5 receptors is a crucial structural determinant for ligand affinity and selectivity
Authors: D'Aoust Jean‐Philippe , Tiberi Mario
Published: June 2021

Human D1‐like dopaminergic G protein‐coupled receptors (D1R and D5R) exhibit marked differences in their ligand binding properties while sharing an overall 80% degree of identity within transmembrane (TM) regions. However, the TM1 regions of D1R and D5R display the most residue differences among all TM regions. Here, we explore the role of TM1 in regulating subtype‐specific ligand binding properties of D1R and D5R. TM1 region of D1R and D5R was swapped using polymerase chain reaction. Binding studies performed in transiently transfected cells show that the D5 chimera has a higher affinity for synthetic drugs as compared with wild type D5R whereas the D1 chimera displays a lower affinity for synthetic drugs in comparison with wild type D1R. Notably, the affinity of the natural ligand dopamine was not changed. Moreover, the selectivity ratio of chimeras for synthetic agonists was increased as compared to that of wild type receptors. Interestingly, the selectivity ratio measured for inverse agonists suggests that in striking contrast to wild type receptors, chimeras bind to inverse agonists with a similar affinity. Overall, our studies highlight a pivotal role of TM1 in shaping the binding pocket for synthetic drugs as well as in regulating differentially the selectivity of D1‐like receptors for synthetic drugs in an efficacy‐dependent manner. Supported by NSERC (#203694‐07; MT) and CIHR Graduate Scholarship (JPD).
Trafficking of System xc‐ is Affected by Cell Culture Density
Authors: Henderson Tara , Wixson Matthew , Sherburn Stephanie , Chase Leah
Published: June 2021

System xc− is a transport system that exchanges extracellular cystine for intracellular glutamate. The transporter plays a role in glutathione synthesis and maintains redox balance within the cell. Thus, the objective of this study was to examine how cellular growth affects System xc− expression and cellular localization in glioma cells. We report that as cell density increases, System xc− activity decreases. Using cell surface biotinylation assays, we showed that the decrease in transporter activity in dense cultures is a result of a loss of membrane localization, not a decrease in overall transporter expression. Upon exposure of dense cells to oxidative agents, more transporters are delivered to the membrane, resulting in equivalent activity to that observed in less dense cultures. However, exposure of less dense cultures to hydrogen peroxide results in a decrease in transporter activity and a formation of a higher molecular weight complex upon exposure to hydrogen peroxide. These data suggest membrane localization is already maximal in less dense cultures and modification of the transporter results in decreased activity. Thus, transporter activity is regulated by oxidative agents at the level of protein synthesis, membrane trafficking, and protein modification. This research was supported by the Campbell Foundation, the NSF REU and MRI programs, and the Biology and Chemistry Departments at Hope College.
Loss of the inactive phosphatase Mtmr13 leads to a Charcot‐Marie‐Tooth type 4B2‐like peripheral neuropathy in mice
Authors: Robinson Fred L. , Niesman Ingrid R. , Beiswenger Kristina K. , Dixon Jack E.
Published: June 2021

Charcot‐Marie‐Tooth disease type 4B (CMT4B) is a demyelinating peripheral neuropathy characterized by slowed nerve conduction velocity (NCV), severe axonal loss, and myelin outfolding and infolding. CMT4B is caused by recessive mutations in either myotubularin‐related protein 2 (MTMR2; CMT4B1) or MTMR13 (CMT4B2). Myotubularins are a large family of phosphoinositide (PI) 3‐phosphatases that dephosphorylate phosphatidylinositol 3‐phosphate (PtdIns3P) and PtdIns(3,5)P2, two PIs that regulate membrane traffic within the endosomal‐lysosomal system. Intriguingly, nearly half of the metazoan myotubularins are catalytically inactive. MTMR2 and MTMR13 are active and inactive PI 3‐phosphatases, respectively, and the two proteins have been shown to directly associate, although the functional significance of this association is not well understood. In hopes of establishing a mouse model of CMT4B2, we have disrupted the Mtmr13 gene. Mtmr13‐deficient mice develop a peripheral neuropathy characterized by reduced NCV and myelin outfolding and infolding. Notably, axonal loss and hypomyelination, two prominent features CMT4B2, are largely not observed in Mtmr13‐deficient mice. Thus, loss of Mtmr13 in mice leads to a dysmyelinating peripheral neuropathy with many of the key features of CMT4B2. Our mouse model will likely be an essential tool for future studies of how the loss of MTMR13 leads to CMT4B2.
Neuregulin 1β/erbB signalling in cardiac myocytes enhances integrin based myocyte‐matrix interactions via src
Authors: Pentassuglia Laura , Arteaga Carlos , Sawyer Douglas
Published: June 2021

The Neuregulin‐1β (Nrg)‐erbB2/ErbB4 (E2/E4) system is critical for the heart and it is needed for myocyte survival in stress conditions. Nrg activates Src and FAK, both part of integrin signaling. Hyp.: Nrg signaling regulates myocyte matrix interactions, critical for coupling force generation to the extracellular matrix.

Immunostaining of mouse hearts showed E4 and E2 are at intercalated discs and have a t‐tubule like striated pattern. Isolated myocytes were incubated 30 min or 24h with an E2 monoclonal antibody (Ab) or an E2 tyrosine kinase inhibitor (TKI). Western‐blot showed E2 TKI blocks activation of FAK, Erk1/2, and Akt by Nrg; Ab is a weak inhibitor. ErbB activation in myocyte‐matrix coupling was tested by plating myocytes onto untreated culture dishes spot‐coated with laminin, with or without Nrg (10ng/ml). After 30 minutes unattached cells were stained and counted. Attachment was enhanced 4‐fold by Nrg vs. laminin alone. Cells did not attach to Nrg coated plates in absence of laminin. Pre‐treatment of myocytes with E2 inhibitors did not prevent Nrg‐enhanced attachment; pre‐treated with Src inhibitor PP2 (10 μM) Nrg effect was inhibited.

Nrg‐1β treatment of cardiac myocytes enhances integrin mediated myocyte‐matrix coupling via Src dependent signalling. This appears to be E2 independent pathway, involving E4 signaling, supported by e4 localization to the intercalated disks in vivo.

Support by SNSF, NIH.
Intramolecular Disulfide Bridges of the Prolactin Receptor (PRLR) Short Form are Required for its Inhibitory Action on the Function of the Long form of the Receptor
Authors: Xie Yili , Hassan Sergio A , Qazi Aamer M , Tsai‐Morris C. H. , Dufau Maria L.
Published: June 2021

The short form (SF), S1b, of the PRLR can silence PRL‐induced activation of gene transcription by the long form (LF). This results from LF/SF heterodimerization and the absence of cytoplasmic (C) sequences in the SF partner, which are essential for STAT activation. Dimerization of PRLR variants is ligand‐independent and PRL is a conformational modifier inducing JAK/STAT signaling. Mutagenesis of SF's four conserved Cys (S1bX), which form two intramolecular S‐S bonds within the extracellular (EC) subdomain‐1 (D1) PRL binding region, eliminated its inhibitory action on LF and its PRL‐induced JAK phosphorylation (JAK‐P). Since constitutive JAK‐P was observed in WT S1b, this change suggests that S‐S provides the conformation for JAK binding to box‐1 (adjacent to the trans‐membrane domain) in the C domain. BRET50 showed decreased heterodimeric association of LF/S1bX and increased affinity in homodimerization of S1bX, which favors LF dimerization and PRL‐induced signaling. Computer simulations of the EC based on the X‐ray crystal structure of PRLR showed less bending of D1 in S1bX. A positive grove that could be a motif of ligand recognition was occluded in S1bX. Additional inter‐monomer hydrogen bonds observed in D1 of S1bX may cause groove occlusion and higher homodimerization affinity of S1bx. The conformation of the PRLR provided by S‐S bridges is essential for the inhibitory action of SF.
Immunolocalization of a sugar transporter involved in sexual development in the fungus Schizophyllum commune by means of epitope‐tagging
Authors: Gallagher Michael Paul , Horton J. Stephen
Published: June 2021

The protein Frt1 has been shown to regulate mushroom development in the common wood‐rotting fungus Schizophyllum commune. Results from a two‐hybrid genetic screen suggest that Frt1 interacts in yeast cells with a putative sugar transporter from S. commune called Sts1. The present study was designed to test if these two proteins interact in the hyphal cells of S. commune, utilizing the techniques of epitope‐tagging and immunofluorescence. A triple HA‐tagged Frt1 protein has already been produced in our lab. A PCR‐based method called splicing by overlap extension (SOEing) was utilized to create a FLAG‐tagged Sts1 protein. The FLAG‐tag was introduced separately into Sts1 at two separate locations, corresponding to the first and third extracellular domains. FLAG‐Sts1 will be transfected into strains of S. commune, which will than be crossed with our HA‐Frt1‐tagged strains. Progeny containing both tagged proteins will be selected, and then examined using the techniques of immunofluorescence and immunoprecipitation for evidence of protein‐protein interactions. Supported by a Union College IEF grant to M.P.G.
Insights into the structure and function of the extracellular domain of alpha‐neurexin by single particle EM and surface plasmon resonance
Authors: Miller Meghan Thorne , Comoletti Davide , Wilson Jennifer , Nakagawa Teru , Taylor Palmer
Published: June 2021

Neurexins are a family of presynaptic cell‐adhesion membrane proteins that are important for synapse maintenance through contact with the postsynaptic neuroligin molecules. Several studies have indicated that mutations in these genes may play an important role in autism and mental retardation. Each neurexin gene contains two promoters corresponding to the α‐and β‐neurexins. While the extracellular domain of β ‐neurexin contains only a single LNS/LG domain, α ‐neurexin consists of six LNS/LG domains, including the β ‐neurexin domain, separated by three EGF domains. To investigate the conformation of the extracellular domain of α ‐neurexin we imaged purified protein using negative‐stain EM. Raw particle EM images suggest that the LNS/LG domains of α ‐neurexin exist in a mixture of different conformations. Additional experiments to obtain class averages and to investigate the effect of calcium and ligands on conformation are ongoing. Using surface plasmon resonance (SPR) we report a rank order of specificity for α ‐neurexin binding to the different neuroligins. Furthermore, we are using mutagenesis and SPR to help us understand the contribution of the different LNS/LG domains of α‐neurexin to neuroligin binding.

This work was supported by: USPHS Grant R37 GM‐18360 and NIEHS to PT, and the Pharmacology Training Grant
Neudesin, a Neurotrophic Factor, Is a Novel Extracellular Heme‐binding Protein.
Authors: Kimura Ikuo , Nakayama Yoshiaki , Mori Masahiro , Ohta Mitsuhiro , Itoh Nobuyuki , Fujimoto Masafumi
Published: June 2021

Neudesin is a secreted protein with neurotrophic activity in neurons and undifferentiated neural cells. We report here that neudesin is an extracellular heme‐binding protein and that its neurotrophic activity is dependent on the binding of heme to its cytochrome b5‐like heme/steroid‐binding domain. At first, we found that at least a portion of the purified recombinant neudesin appeared to bind hemin because the purified neudesin solution was tinged with green and had a sharp absorbance peak at 402nm. The addition of exogenous hemin extensively increased the amount of hemin‐bound neudesin. In contrast, neudesinΔHBD, a mutant lacking the heme‐binding domain, could not bind hemin. The neurotrophic activity of the recombinant neudesin that bound exogenous hemin (neudesin‐hemin) was significantly greater than that of the recombinant neudesin in either primary cultured neurons or Neuro2a cells, suggesting that the activity of neudesin depends on hemin. The inhibition of endogenous neudesin by RNA interference significantly decreased cell survival in Neuro2a cells. This indicates that endogenous neudesin possibly contains hemin. The experiment with anti‐neudesin antibody suggested that the endogenous neudesin detected in the culture medium of Neuro2a cells was associated with hemin because it was not retained on a heme‐affinity column at all. Neudesin is the first extracellular heme‐binding protein that shows signal transducing activity by itself. The present findings may shed new light on the function of extracellular heme‐binding proteins.
The Effects of Cell Surface NgCAM Expression on Developing Chick Brain Axons In Vitro
Authors: Culver Stephanie , Galileo Deni S
Published: June 2021

During embryonic brain development, molecular guidance cues work in concert to transduce signals to guide axons to their appropriate targets. Of these cues are proteins of the Ig superfamily, including L1 and its chick homologue, neuron‐glia cell adhesion molecule (NgCAM). L1 and NgCAM are neuronal cell surface membrane proteins shown by others to accelerate axonogenesis and increase axon elongation. In vitro bioassays were performed in order to test the hypothesis that NgCAM expression on the cell monolayer will increase rate of outgrowth, axon length, and number of axon processes. Embryonic Day 7 optic tectum (OT) explants were first used to study outgrowth, yet failed to be an efficient and reproducible model. Aggregates were then made from dissociated OT cells and plated onto NIH3T3 monolayers that did or did not express uniform NgCAM and onto monolayers exhibiting patterned NgCAM expression. The aggregates were visualized using static images and time‐lapse microscopy by fluorescent membrane dye. Current data has shown an increase in both axon length and number in the presence of NgCAM, suggesting its crucial role as a positive axon guidance cue. Supported in part by HHMI and NIH.
Characterization of direct ERK‐mediated activation of the Dopamine Transporter
Authors: Schafer Marissa , Remmick Jennifer , Brandvold Kristopher , Foster James , Vaughn Roxanne , Provost Joseph E
Published: June 2021

Primarily localized in the substantia nigra and tegmental areas of animal brain tissue, the neurotransmitter dopamine (DA) mediates numerous CNS functions including motor activity, emotion, and reward. Synaptic and extracellular DA concentrations are controlled by the dopamine transporter (DAT), which is activated or down‐regulated in response to both allosteric and feedback mechanisms including protein kinase pathways and dopamine receptor feedback signals from adjacent neuronal membrane surfaces. Many mechanisms that play a role in DAT regulation have been characterized, such as PKC‐dependent DAT trafficking. However, physiological DAT activity is likely to be mediated by other Ser/Thr‐kinases, such as ERK. For this study, polyclonal antibodies against ERK phosphorylated rDAT N‐terminus were raised against the N‐terminus tail of recombinant DAT and phospho‐DAT peptides. To examine the effect of ERK on DAT activation, ERK phosphorylation was consistently reduced in rDAT‐LLC‐PK cells through the application of specifically defined culture conditions including optimum confluency, media, and culture serum starvation length. To verify phospho‐ERK levels, control and stimulated rDAT‐LLC‐PK lysate was prepared and subjected to immunoanalysis by p‐rDAT antibodies. Follow‐on studies investigated the effects of expressing dominant negative MEK on DAT activity and ERK phosphorylation of rDAT. These studies indicate the involvement of ERK in DA metabolism and suggest a novel role for ERK in CNS functions.
Characterization of GCN5‐Containing Complexes in Human Cells
Authors: Wang Yuan‐Liang , Faiola Francesco , Pan Songqin , Martinez Ernest
Published: June 2021

The packaging of DNA into chromatin by histones and non‐histone proteins creates substantial opportunities for the regulation of DNA replication, repair, and transcription. Eukaryotic cells possess numerous large multi‐protein complexes that control the functions of the genome by modifying chromatin structure. One of the most studied histone modification is the acetylation of specific lysine residues on histone tails. To date, a large number of histone acetyltransferases (HATs) have been identified, and GCN5 is the first prototypical HAT to show a link to transcription activation. It functions as the enzymatic subunit within multi‐protein coactivator complexes (e.g. the yeast SAGA and human STAGA/PCAF complexes) to regulate chromatin structure and gene transcription. We have shown recently that, in addition to its HAT function, the human SAGA‐like complex STAGA directly recruits Mediator to activate transcription. We have now identified novel subunits of the STAGA complex. Furthermore, we have found that GCN5 (and its paralog PCAF) stably associates with additional proteins in human cells and forms a novel 1.8 MDa multi‐protein complex that is structurally and functionally distinct from STAGA/PCAF.
The cell cycle inhibitor p21(WAF1/Cip1) as a transcriptional regulator of pigment cell‐specific gene expression
Authors: Vachtenheim Jiri , Sestakova Blanka
Published: June 2021

The cyclin dependent kinase inhibitor p21(WAF1/Cip1) has been shown previously to regulate expression of a number of genes related to cell cycle regulation, DNA repair, senescence, differentiation and apoptosis. Recently, transcription of p21 was found to be activated by MITF (microphthalmia‐associated transcription factor), a factor which plays a central role in melanocyte‐specific transcription. Here we investigated the expression and transcriptional activity of p21 in human melanoma cells. We found no correlation between proliferation and p21 protein level in unsynchronized melanoma cells. p21 protein levels were surprisingly higher in low‐pigmented, faster proliferating normal melanocytes than in dark‐pigmented slowly proliferating melanocytes. Furthermore, clones of melanoma cells with p21 level stably knocked‐down by siRNA had even decreased growth rates when compared to controls. We further found that promoters of several genes related to differentiation were stimulated by p21 overexpression, including the MITF promoter. Mutational analysis of p21 suggested that mechanisms other than cell cycle inhibition, PCNA binding, and derepression of p300/CBP repression domain CRD1 underlie the transcriptional effects. These results suggest that p21 may have a role in transcription rather than in cell cycle regulation in melanoma cells.

Grant support: MZO00064211, NR/9319‐3, MH, Czech Rep.
DNA Topoisomerase I and DNA Topoisomerase II Are Regulated Post‐translationally in Interleukin‐2 Activated HuT 78 Cells
Authors: Foglesong Paul David , Martinez Adela M. , Gomez John Eric
Published: June 2021

Interleukin‐2 (IL‐2) is a lymphokine that binds to a high affinity receptor (CD 25) on the surface of human T lymphocytes. Transduction of this signal activates T cells by means of biochemical pathways that result in increased transcription, DNA replication, and, ultimately, cell division. HuT 78, a CD 25+ human T cell lymphoma line, was obtained from ATCC and used in these studies. It was previously shown that the specific activities of both DNA topoisomerase I (Topo I) and DNA topoisomerase II (Topo II) increased 10‐fold to 20‐fold in nuclear extracts prepared from HuT 78 cells within the first 24 hrs. after treatment with IL‐2 (Foglesong, 2006). Those results suggested that both Topo I and Topo II function in transcription and DNA replication in activated human T lymphocytes. The polypeptide composition of the nuclear extracts was analyzed by SDS‐polyacrylamide gel electrophoresis in order to determine whether the observed increases in the specific activities of these enzymes were due to increased synthesis of protein. Aliquots of the nuclear extracts containing 1–5 μg protein were incubated at 100o C for 2 min. in SDS buffer and electrophoresed at 75 V for 2 hr. on 4 to 20% gradient polyacrylamide gels along with broad range markers (Bio‐Rad). The gels were stained with Coomassie Brilliant Blue. Under these denaturing conditions the relative molecular masses (Mr) for Topo I, Topo IIα, and Topo IIβ are 100, 170, and 180 kDa, respectively. No changes in the abundance of proteins of these sizes were observed in the extracts prepared in the first 24 hrs. following treatment of HuT 78 cells with IL‐2. These results taken together suggest that the activities of Topo I and Topo II are regulated post‐translationally in IL‐2‐activated human T lymphocytes. This work was supported by CA 74388 to PDF.
TRPC‐1 knockdown abrogates nuclear factor‐kB‐dependent anti‐apoptotic gene expression induced by thrombin in endothelial cells
Authors: Thippegowda Prabhakar Bettadathunga , Tiruppathi Chinnaswamy , Xue Jiaping , Sundivakkam Premanand , Singh Vandna , Malik Asrar B
Published: June 2021

Thrombin activation of protease‐activated receptor‐1 induces Ca2+ influx through store‐operated Ca2+ entry channels (SOC) in endothelial cells. Previously we have shown that the canonical TRPC isoform TRPC1 is an essential component of SOC in endothelial cells. Further, we showed that thrombin‐induced Ca2+ influx signal activates nuclear factor‐kB (NF‐kB) in endothelial cells. In this study we measured thrombin‐induced expression of NF‐kB regulated genes in human pulmonary artery endothelial cells (ECs) utilizing Gene Microarray. Thrombin stimulation increased at least sixteen genes between 5‐ and 20‐fold over basal in ECs. Thrombin‐induced expression of anti‐apoptotic genes such as A20, IL8, and BCL3 were markedly reduced by pharmacological inhibition of Ca2+ influx in ECs. TRPC1 knockdown with siRNA abolished thrombin‐induced Ca2+ influx and markedly reduced A20, IL8, and BCL3 expression in ECs. Thus our results suggest that Ca2+ influx via SOC activates NF‐kB signaling to induce expression of A20, IL8, and BCL3 to promote cytoprotective effect in endothelial cells.
Absence of Nitric Oxide Synthase in Exquisitely Pure Rat Liver Mitochondria
Authors: Venkatakrishnan Priya , Nakayasu Ernesto S. , Almeida Igor C. , Miller Richard T.
Published: June 2021

The nitric oxide synthases (NOS) catalyze the conversion of L‐arginine to L‐citrulline plus NO. Data, both for and against the presence of a mitochondrial NOS (mtNOS) isoform, is in the refereed literature base. Irrefutable evidence, however, for a mtNOS isoform has not been forthcoming and conflicting reports continue to be published. In light of this controversy, we designed meticulous studies to confirm/refute the existence of mtNOS. Pure, rat liver mitochondria were obtained by differential centrifugation followed by Percoll purification. Positive controls contained mitochondria spiked with purified, recombinant nNOS (nNOSr). Analysis of sub‐mitochondrial particles by LC‐MS failed to detect any NOS isoform in mitochondria whereas nNOSr peptides in spiked samples as low as 50 fmol were detected. [C14] L‐arginine to [C14] L‐citrulline conversion was negative for NOS activity. Additionally, detection of NO production by mitochondria by use of the oxyhemoglobin capture assay run with or without SOD & catalase were also negative. Moreover, Western blot analyses using nNOS & eNOS antibodies failed to detect these two isoforms in mitochondria. In conclusion, by avoiding previously published errors in methodologies and sample preparation, and by using correct controls, we refute the claim that a NOS isoform exists within rat liver mitochondria. (Supported by ES011982 & RR008124 to RTM & UTEP, respectively)
A Novel nNOS Signaling Pathway Regulates Skeletal Muscle Size, Strength and Fatigue Resistance
Authors: Percival Justin , Anderson Kendra , Huang Paul , Froehner Stanley
Published: June 2021

Nitric oxide (NO) is a highly versatile signaling molecule which participates in a broad array of pathways in the cardiovascular, immune and nervous systems. We present further evidence for this physiological diversity by identifying a novel nNOS signaling pathway in skeletal muscle. NO is synthesized by neuronal nitric oxide synthase mu isoenzyme (nNOSμ) in skeletal muscle. nNOSμ is found associated with the dystrophin‐associated glycoprotein complex and in a soluble pool. During investigation of the soluble nNOS pool, we identified a splice variant of nNOS called nNOSβ localized to the Golgi complex. Soluble guanylyl cyclase and protein kinase G also localized to the Golgi suggesting that a classical NO–cGMP signaling pathway functioned at the Golgi complex. Loss of nNOSβ activity resulted in reduced muscle mass with muscles exhibiting lower force‐generating capacity, even after correcting for decreased muscle size. Furthermore, the absence of nNOS decreased the ability of muscle to resist exercise‐induced fatigue. In summary, we have identified a novel nNOS‐signaling pathway that regulates skeletal muscle size and strength and provided the first evidence that differential targeting of nNOS isozymes facilitates the creation of spatially and functionally distinct nNOS signaling pathways by localized synthesis of an otherwise directionless free radical gas. Furthermore, these results make nNOSβ of potential relevance to myopathies and dystrophies where preventing the loss of muscle bulk and strength is of clinical importance.
Cilostazol reduces prolifferation in MDCK cells through down‐regulation of c‐Myc
Authors: Muñoz Rodrigo , López‐Bayghen Esther
Published: June 2021

Polycystic kidneys are inherited diseases characterized by excessive proliferation and apoptosis of renal tubular epithelia, cysts development and progressive renal insufficiency. Emerging therapies are c‐myc antisense oligonucleotides or phosphodiesterases (PDEs) inhibitors. Cilostazol is a specific inhibitor of PDE3, used as antithrombotic and antiplatelet agent because inhibits vascular cell proliferation increasing cAMP levels, downregulating E2F activity and inhibiting cell cycle associated kinases.

Treatment of MDCK cells (cystogenesis model) with Cilostazol results in decrease of proliferation rates and downregulation of transcription factor c‐Myc. Reduced expression and DNA/binding were observed. Decreased transcriptional activity of E2F and cyclin D1, promoters with E‐Boxes was recorded. In contrast DNA/binding and activity of the differentiation related factor Sox10 increased. Using cytometry flow analysis we found arrest in G1/S phase and apoptosis; DAPI staining analysis showed that this drug does not cause complete DNA damage. Response to Cilostazol is probably mediated by an increase in cAMP, PKA activation and inhibition of ERK. Results indicate that cilostazol downregulates c‐Myc and other elements controlling cell cycle progression in MDCK, reducing proliferation rates, activity that may contribute to kidney cyst treatment.

Support: CONACyT 50414
Effect of estrogen and progesterone on the distribution and activity of thimet oligopeptidase in rat and mouse brain
Authors: Kua Lindsay H. , Cyr Nicole E. , Tetel Marc J. , Chadwick Joe G. , Bruce Lisa A. , Wolfson Adele J.
Published: June 2021

Thimet oligopeptidase (TOP) is a soluble metalloendopeptidase that naturally degrades gonadotropin‐releasing hormone (GnRH), a key player in the hypothalamic‐pituitary‐gonadal axis of the body. GnRH controls the production of estrogen and progesterone, high levels of which in turn inhibit the release of GnRH from the hypothalamus. We hypothesize that TOP is involved in this feedback loop, specifically that it is upregulated or activated by high levels of estrogen to aid in the removal of GnRH. Previous studies have focused on enzyme activity in regions of the rat brain, whereas we have used both rats and mice to study how levels of TOP, as assessed by western blot and other immunological methods, change in response to ovariectomy and subsequent estrogen and/or progesterone replacement. Ovariectomized animals each received one of three treatments: oil, estrogen, or estrogen followed by progesterone. TOP levels in rats were highest in the hypothalamus compared to the hippocampus, amygdala and thalamus. Similarly, TOP levels were greater in mouse hypothalamus than hippocampus. Among the three treatment groups, estrogen treatment was correlated with the highest levels of TOP and TOP enzymatic activity in the rat hypothalamus. Our findings indicate that TOP may be upregulated by estrogen in the hypothalamus in order to break down GnRH once it is secreted.
Identification of regions of bacterial topoisomerase I important for cleavage‐religation for the discovery of type IA topoisomerase poisons as novel antibacterial compounds
Authors: Tse‐Dinh Yuk‐Ching , Cheng Bokun , Abrenica Maria V , Sorokin Elena P , Wong Agnes
Published: June 2021

Type IA and type IIA topoisomerases have little sequence homology but similar mechanisms of action. We are targeting bacterial type IA topoisomerases in our HTS efforts to obtain new leads for discovery of novel antibacterial compounds to combat multi‐drug resistant bacterial pathogens. The goal is to identify small molecules that can act as poisons of bacterial type IA topoisomerases by trapping the covalent intermediate formed between the enzyme and cleaved DNA, similar to the action of fluoroquinolones on bacterial type IIA topoisomerases. We have previously demonstrated that expression of recombinant type IA topoisomerases with the strictly conserved glycine residue in the DXDXXG TOPRIM motif mutated to serine led to accumulation of the covalent cleaved DNA complex and rapid bacterial cell killing. This validates type IA topoisomerases as potential targets for discovery of novel antibacterial drugs acting as type IA topoisomerase poisons. We have isolated additional SOS‐inducing Yersinia pestis and Escherichia coli topoisomerase I mutants and characterized these mutant enzymes biochemically. These mutagenesis results identify regions of the enzyme important for control of the cleavage‐religation equilibrium for potential docking of small molecules to achieve similar bactericidal action as the topoisomerase I mutations.
Identification and characterization of bacterial genes conferring resistance to cell killing by topoisomerase‐mediated DNA cleavage
Authors: Liu I‐Fen , Tse‐Dinh Yuk‐Ching
Published: June 2021

Antibiotics therapy is based on the distinct class‐specific drug‐target interactions, but they may also exert similar bactericidal mechanism via toxic side products. We are investigating the molecular regulatory mechanism of bacterial resistance action in conjunction with high throughput screening of new antibacterial agents acting as bacterial type IA topoisomerase poisons. Gene amplification is often a mechanism for bacteria to overcome challenges to survival. The major goal of this study is to identify genes of E. coli, which when present in high copy number, can suppress the type IA and IIA bacterial DNA topoisomerase poisons, and to examine their molecular mechanism of regulating cell death mediated by trapped topoisomerase cleavage complexes. In this study, the E. coli strain (BW27784‐pAYTOP128) expressing SOS‐inducing and cell killing mutant of Y. pestis topoisomerase I was used as a model system to screen for such genes from a genomic library present on high copy number plasmid. We have isolated several plasmid clones that can confer resistance to the mutant topoisomerase I‐mediated cell killing. These genes are being tested to determine if they also contribute to quinolone antibiotics resistance. Resistance to other bactericidal antibiotics which do not accumulate topoisomerase cleavage complex but induce the formation of ROS is also being studied to determine the mechanism of resistance.
Distinct and Overlapping Functions for Thymic Stromal Lymphopoietin‐ and Interleukin 7‐driven B cells in T cell‐independent Antibody Responses
Authors: Shriner Anne K. , Sun Guizhi , Leonard Warren , Alugupalli Kishore R.
Published: June 2021

T cell independent (TI) responses are impaired in young children but not in adults. B1b cells mount the majority of the TI antibody response to pneumococcal polysaccharide and Borrelia hermsii. Although antigens driving this response have been identified, the developmental origin of the antigen‐specific B1b cells is unknown. Thymic stromal lymphopoietin (TSLP) is responsible for most perinatal B cell development, while interleukin 7 (IL7) plays a crucial role in adult B lymphopoiesis. To determine whether distinct B1b cell development pathways are accountable for the impaired TI responses in young children, we investigated antibody responses to a model TI antigen, NP‐ficoll, as well as to B. hermsii infection in mice deficient in either IL7, the TSLP receptor or IL7Rα, a subunit required for both IL7 and TSLP signaling. TSLPR−/− mice exhibited a normal response to NP‐Ficoll suggesting that IL7‐driven B1b cells constitute the majority of antibody production. However, IL7−/− mice controlled B. hermsii bacteremia as efficiently as TSLPR−/− or wildtype mice suggesting the generation of functionally redundant B1b cells. Indeed, IL7Rα−/− mice were severely impaired in their response to B. hermsii as well as NP‐Ficoll. These data indicate distinct and overlapping roles for IL7‐ and TSLP‐driven B1b cells in TI immunity and suggest that impaired responses in the young are due to an insufficient IL7‐driven B cell response.
Interleukin (IL)‐7 promotes T‐cell survival by stabilization of Mcl‐1
Authors: Li Wenqing , Hixon Julie , Durum Scott
Published: June 2021

IL‐7 is required for T‐cell development both in mouse and humans and for T‐cell homeostasis in the periphery. Some of the downstream signal transducers from the IL‐7 receptor are members of the Bcl‐2 family of proteins associated with mitochondrial apoptosis. Mcl‐1, one of the anti‐apoptotic members of Bcl‐2 family, became of particular interest when it was discovered that IL‐7 upregulates Mcl‐1 mRNA expression in T cells. This suggests that Mcl‐1 may contribute to the survival functions of IL‐7. In this study, we showed that the deficiency of T–cell development in IL‐7Rα KO mice could be rescued by reconstitution of IL‐7Rα deficient bone marrow by retrovirus expressing a mouse Mcl‐1 construct. To further investigate the molecular mechanism of IL‐7 regulation of Mcl‐1, we used an IL‐7‐dependent murine thymic cell line D1. Deprivation of IL‐7 from D1 cells or lymph node cells caused Mcl‐1 protein degradation by 6 hrs. Overexpression of Mcl‐1 protected D1 cells from the death induced by IL‐7 deprivation. Compared to intact Mcl‐1, the truncated form of Mcl‐1 (Δ375), with deletion of 375 nucleotides from its 5′end, showed more potent anti‐apoptotic activity, suggesting the 5′end of Mcl‐1 might contain regulatory domains. In human Mcl‐1 the lysine in the 5′ end of the gene was identified as the target site for ubiquitin. Based on this finding, we hypothesized that the lysine found in the mouse Mcl‐1 would share this function. To test this hypothesis, we mutated the first three lysine residues (39, 80, and 117) to alanine at mouse Mcl‐1 (Mcl 3KR) and tested its protein stability and its ability to prevent apoptosis. Mcl 3KR showed enhanced protein stability, and also provided D1 cells with more protection, compared to WT Mcl‐1, after 3 to 4 days of IL‐7 deprivation. Our findings suggest that Mcl‐1 plays an important role protection of T‐cell survival by IL‐7. IL‐7 strongly promotes Mcl‐1 stability, possible by controlling lysine‐directed ubiquitination.
Differential Requirement for Nuclear Factor TOX in CD4 and CD8 T Cell Lineage Development in the Thymus
Authors: Aliahmad Parinaz , Kaye Jonathan
Published: June 2021

We have previously identified a nuclear factor member of the HMG‐box superfamily, designated TOX (thymocyte selection‐associated HMG box protein), which is transiently expressed in the thymus during both positive selection and β‐selection. We now report that mice deficient in TOX (Tox−/−) have severe reductions in all CD4 lineage T cells, including conventional T, regulatory T and NK‐T cells, due to a block in the CD4loCD8lo to CD4+CD8lo thymocyte transition of positive selection. We did not detect a TCR signaling defect in Tox−/− thymocytes and loss of the CD4 T cell lineage is not due to increased cell death, as expression of the anti‐apoptotic protein BCL‐2 does not rescue the development of CD4 T cells. The loss of the CD4 lineage, however, is associated with failure to upregulate Zbtb7b, but not GATA3, during positive selection. In contrast, functional CD8 T cells can develop in Tox−/− mice. The block in development of CD4 but not CD8 T cells in Tox−/− mice is also observed in TCR transgenic animals. Moreover, the failure of class II MHC restricted T cells to be misdirected into the CD8 lineage in these mutant mice has implications for the mechanism of lineage commitment in the thymus. These results demonstrate differential requirements for the CD4 and CD8 lineages, where CD4‐destined cells require induction of TOX and passage through a CD4+CD8lo stage for their development, while CD8‐destined cells can develop directly from CD4loCD8lo thymocytes. We are currently working to identify the changes in gene expression mediated by TOX.
Role of MAPK Pathway in Cell Differentiation and Cell Proliferation in Early Hematopoietic Progenitors
Authors: Chung Eva , Hsu Chia‐lin , Kondo Motonari
Published: June 2021

During hematopoietic development, hematopoietic stem cells (HSCs) commit to either the myeloid or lymphoid lineage. Recently, we have shown that the mitogen activated protein kinase (MAPK) pathway is involved in granulocyte/macrophage (GM) lineage commitment under physiological conditions. MAPK kinase (MEK) inhibitors resulted in reduced numbers of granulocyte/macrophage (GM) colonies from HSCs. In addition, the MAPK pathway has been implicated in many types of cancers, including leukemias and lymphomas. Specifically, MEK and its downstream target, extracellular signal‐regulated kinase (Erk), have been shown to be constitutively active in some types of human leukemias. While many studies have been done using cell lines transfected with constitutively active MEK or Erk, no such studies have been conducted with primary cells. In this study, we introduced the constitutively active form of MEK into HSCs and injected the cells into lethally irradiated mice. Our data demonstrates that HSCs transduced with constitutively active MEK leads to massive expansion of granulocytes and macrophages, which is characteristic of chronic myelomonocytic leukemia (CMML) in humans. We are currently characterizing the disease and defining the role of the MEK/ERK pathways in malignant transformation in HSCs.
Bmi1 regulates memory Th2 cell survival via repression of the Noxa gene.
Authors: Nakayama Toshinori
Published: June 2021

The maintenance of memory T cells is central to the establishment of immunological memory, although molecular details of the process are poorly understood. In the absence of the Polycomb group (PcG) gene Bmi1, the number of memory Th2 cells was reduced significantly. Enhanced cell death of Bmi1−/− memory Th2 cells was observed both in vivo and in vitro. Among various pro‐apoptotic genes that are regulated by Bmi1, the expression of pro‐apoptotic BH3 only protein Noxa was increased in Bmi1−/− effector CD4 T cells even in the absence of Ink4a and Arf. The generation of memory Th2 cells was restored by the deletion of Noxa but not by Ink4a and Arf. Direct binding of Bmi1 to the Noxa gene locus was accompanied by histone H3‐K27 methylation. The recruitment of other PcG gene products to the Noxa gene was highly dependent on the expression of Bmi1. In addition, DNA methylation appeared to control the recruitment of PcG gene products and the expression levels of Noxa. Moreover, memory Th2‐dependent airway inflammation was attenuated substantially in the absence of Bmi1. Thus, Bmi1 controls memory Th2 cell survival and function through the direct repression of the Noxa gene.