bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of GIV‐Gαi3, a Non‐Receptor GEF‐G protein complex involved in cell migration and metastasis
Authors: Garcia‐Marcos Mikel , Ghosh Pradipta , Ear Jason , Farquhar Marilyn Gist 
Published: June 2021

G‐protein coupled receptors (GPCRs) are the canonical guanine nucleotide exchange factors (GEFs) that activate trimeric G proteins. In contrast to GPCRs, little is known about the structure or function of non‐receptor GEFs. We previously reported that GIV/Girdin is a non‐receptor GEF for Gαi‐subunits that promotes cell migration and tumor cell metastasis (PNAS, 2009, 106: 3178–83). We discovered an evolutionarily conserved GEF motif in GIV that forms a unique interface with the Gα‐subunit. Mutation of a key residue (F1685) within this motif disrupted the Gαi‐GIV interaction and made GIV GEF‐deficient. We have now extensively characterized the molecular basis for the interaction between GIV and Gαi3 and its functional consequences. We found that GIV and Gαi3 can form a stable 1:1 complex in which the rate of nucleotide exchange, but not GTP hydrolysis, by the G protein is enhanced. Based on the preferential binding of GIV to Gαi versus Gαo, we have identified W258 in the Gαi‐subunit as a critical determinant for GIV binding. Mutation of W258 to the corresponding F in Gαo rendered Gαi3 GEF‐insensitive but did not perturb interaction with other Gα binding partners, i.e., Gβγ, GPCRs, GDIs or GAPs. When either the GEF‐deficient GIV mutant F1685A or GIV‐insensitive Gαi3 mutant W258F were expressed in HeLa cells they failed to undergo cell migration and to promote Akt signaling. These findings provide valuable information on the structural and biochemical basis for the biological functions of GIV as a Gαi activator which may be useful in the design of small molecules to disrupt the Gαi‐GIV interface for therapeutic purposes.Support: NIH DKI7780 and CA100768 (M.G.F). Susan G. Komen KG080079 (M.G‐M). NIH/T32 DK07202 and RSA from American Gastroenterology Association (P.G). McNair Student Research Scholarship (JE).
Tributyltin causes activation of the MAP3K, c‐Raf, but not Ras in human natural killer cells
Authors: Celada Lindsay , Whalen Margaret 
Published: June 2021

The toxic chemical tributyltin (TBT) has been found in human blood. TBT is employed in various industrial applications and due to these uses contaminates the environment. Human natural killer (NK) cells are a subset of lymphocytes capable of destroying tumor cells, virally infected cells, and antibody‐coated cells. It has been shown that TBT is able to diminish the lytic function of NK cells. Previous studies have demonstrated that exposure to TBT leads to the activation of Mitogen‐Activated‐Protein‐Kinase (MAPK) and MAPK‐Kinase (MAP2K) in NK cells. TBT also results in the activation of the MAPK Kinase‐Kinase (MAP3K), c‐Raf, at an exposure time of ten minutes in NK cells. Activation/phosphorylation of c‐Raf (Ser338) was seen with exposure to 300, 200, 100, and 50 nM TBT (2 fold at 300–100 nM and 1.45 fold at 50 nM). Increased phosphorylation of Ser259 was also seen with 300–200 nM TBT (2 and 1.7 fold, respectively). Ras is a GTPase which is known to lead to the activation of the MAP3Ks. In its inactive state, Ras is bound to GDP; but when activated, it binds GTP. The focus of this study was to see if there was a TBT‐induced activation of Ras that leads to activation of Raf in NK cells. It was found that exposure to TBT in these cells did not result in the activation of Ras; indicating that there is an alternative mechanism for activation of Raf by TBT.Supported by NIH grant S06 GM008092‐34
PPB is a Novel Serine/Threonine Phosphatase of Akt and is Involved in Myogenesis
Authors: Duan Xueyan , Tao Shen , Gao Xinxin , Zhang Jin , Lin Xia , Feng Xin‐Hua 
Published: June 2021

PI3 kinase/Akt signaling pathway plays a central role in many biological functions. There are two phosphorylation sites on Akt (Thr308 and Ser473), which are phosphorylated by PI3‐kinase/PDK1 and mTOR‐rictor kinase complex, respectively. Recently, PH domain and leucine rich repeat protein phosphatase 1(PHLPP1) was identified to dephosphorylate pSer473. The identity of phosphatase on Thr308 is still elusive.We screened for Akt phosphatase on Ser473 and Thr308 in mammalian cells. Of 45 phosphatases, we identified PPB (Protein Phosphatase for Akt/PKB) as a potent phosphatase that dephosphorylates both Thr308 and Ser473. This was supported by both in vivo and in vitro phosphatase assays. Fluorescence resonance energy transfer (FRET) analysis using AktAR reporter indicates that PPB reduces Akt activity in live cells. However, PPB dead mutant doesn't dephosphorylate Akt. All these data suggest that PPB dephosphorylates Akt and reduces its kinase activity.PI3K/Akt pathway plays an important role during myogenesis. To study the effect of PPB on myogenesis, we generated C2C12 stable cells that over‐express PPB. When C2C12 control and stable cells were differentiated for up to 3 days, the expression of two important myogenetic markers, myosin heavy chain (MHC) and myogenin was abolished, However, this can be rescued with gain‐of‐function Akt, suggesting the inhibition of myogenesis in C2C12 cells by PPB was through its dephosphorylation of Akt.
The nerve growth factor receptor TrkA exists as a pre‐formed homodimer before ligand binding
Authors: Shen Jianying , Maruyama Ichiro 
Published: June 2021

The nerve growth factor (NGF) family of neurotrophins plays a central role in the development, growth, and maintenance of the nervous system. These neurotrophins transduce their effects by interacting with two types of cell surface receptors, the Trk family of tyrosine kinase receptors, and p75NTR. Two classes of NGF‐binding sites, high and low affinities, are present on the surface of neurons. When TrkA and p75NTR receptors are co‐expressed, the association rate is dramatically increased, creating a new site consistent with the NGF high affinity binding site. These results suggest that the receptors interact each other prior to ligand binding.In the present study, we have analyzed the structures of the TrkA receptor for NGF by bimolecular fluorescence complementation and luciferase fragment complementation assays as well as by chemical crosslinking of the receptor expressed on the cell surface. These analyses demonstrated that before ligand binding, the majority of receptors exist as a homodimer in living cells. Using Brefeldin A, a lactone antibiotic that disassembles the Golgi apparatus and block anterograde transport of the receptors from ER to Golgi, furthermore, it has been found that TrkA homodimers are initially formed in ER before reaching to the cell surface. This work provides new insights into understanding of transmembrane signaling by TrkA receptors.
Diacylglycerol production at the Golgi: regulation by Ca2+
Authors: Kunkel Maya , Newton Alexandra 
Published: June 2021

Protein kinase C and protein kinase D are potently activated by agonist‐evoked increases in diacylglycerol. Using live cell‐imaging probes for kinase activity, we have previously shown that both kinases are robustly active at the Golgi following stimulation of Gq‐coupled receptor signaling pathways, displaying activation signatures at the Golgi that are distinct from those at the plasma membrane. Here we address the mechanism by which signals received at the plasma membrane cause these two protein kinases to be activated at the Golgi. Using FRET‐based reporters to image diacylglycerol production, we show that Ca2+ is necessary and sufficient to transduce signals from the plasma membrane to the Golgi. First, chelation of intracellular Ca2+ prevents UTP‐dependent increases in diacylglycerol at the Golgi, but not plasma membrane. Second, raising intracellular Ca2+ by treating cells with thapsigargin induces diacylglycerol production at the Golgi. Thus, agonist‐evoked increases in intracellular Ca2+ cause increases in Golgi diacylglycerol, allowing this intracellular membrane to serve as a platform for signaling by protein kinases C and D.This work was supported by NIH GM43154 and NIH DK54441.
ANALYSIS OF GENETIC VARIATION BETWEEN WILD AND MUTANT STRAINS OF HYPER‐PECTINOLYTIC Aspergillus niger USING RAPD‐ PCR
Authors: OKAFOR UZOMA ANTHONY , Okochi Veronica Ihuaku , Ebuehi O Albert O , Okpuzor Joy , Adekunle Adedotun A 
Published: June 2021

Improving the value of agrowastes generated via fungi degradation has become a priority in most large biomass generating countries for economic and ecological reasons.Wild strain of Aspergillus niger previously isolated from agrowastes in Lagos Nigeria, was subjected to UV‐mutagenesis; through the use of a selective culture medium containing pectin and 2 deoxy D glucose (2dg) to isolate hyper pectinolytic mutant strains. The polymerase chain reaction (PCR) that includes random amplified polymorphic DNA (RAPD) was employed to investigate the influence of UV‐mutagenesis in inducing mutation in the wild strain.Genetic relationship among isolates was determined by pair‐wise comparisons of the shared number of DNA bands on an agarose gel. The results showed that there was considerable genetic variation among isolates. The wild and the mutant strains of the fungal isolates shared an average of only 20% and 42 % of the bands in their RAPD profiles using OPA 2 and OPA 4 respectively, while they shared an average of 60 and 56% of the bands using OPC 3 and 8 respectively. The results indicated that there is genetic variability between the wild and mutant strains; signifying the possibility of the use of UV mutagenesis for generation of hyper‐pectinolytic fungi for effective biodegradation of agrowastes and enzyme production.
The role of methionine cycle metabolites in autoimmune neurodegenerative diseases
Authors: Gardner Lidia A , Hartzes Anastasia M , Clark Daniel , Gotuzzo Eduardo , Levin Michael C 
Published: June 2021

ObjectiveTo compare levels of Homosysteine (Hcy), S‐adenosyl‐methionine (SAM) and Methionine‐Adenosyltransferase‐II (MAT‐II) enzyme activity in patients with multiple sclerosis (MS) and human T‐lymphotrophic virus type 1 (HTLV‐1) associated myelopathy/tropical spastic paraparesis (HAM/TSP).Four groups were evaluated: 1) HAM/TSP; 2) asymptomatic carriers of HTLV‐1; 3) MS and 4) normal age‐matched controls.The mean plasma tHcy levels were higher in HAM/TSP (9.49±1.53μM/L), MS (9.85±1.77μM/L) and the asymptomatic group (12±1.4μMol/L) compared to controls (6.56±0.98μMol/L). The asymptomatic HTLV‐1 infected patients had statistically higher (P=0.0046) tHcy levels compared to the other groups (P=0.0277, Kruskal‐Wallis test). Also, when stratified by age, there was a trend towards significance in Hcy levels comparing diseased (HAM/TSP, MS) to normal controls. Next, we compared MAT‐II activity between MS patients and age‐matched controls. MAT‐II was higher in MS group (Km = 18.84 in MS vs. 12 in controls, P=0.0074). There was no significant difference in SAM (mean range from 10.92 to 12.97μMol) levels between any of the groups. These data underscore the complexity of the methionine cycle and suggest that high tHcy levels may be a marker for viral infection or cause damage to neurons by activation of oxidative injury and DNA damage pathways in autoimmune neurological disease.
Enhanced expression of Glia maturation factor in Alzheimer's disease
Authors: Zaheer Asgar , Zaheer Smita , Thangavel Ramasamy , Ramamoorthy Lavanya , Yang Xi , Sahu Shailendra K 
Published: June 2021

We have previously demonstrated that Glia maturation factor (GMF), a brain‐specific protein isolated, sequenced and cloned in our laboratory, is a prominent mediator of inflammation in CNS leading to the death of neurons. In the present study we demonstrate, for the first time, a significant up regulation of the GMF in AD brain compared to age matched ND (non‐demented) controls. We analyzed brain regions by ELISA using specific anti‐GMF antibodies developed and characterized in our laboratory. We observed a significant increase in GMF concentration in entorhinal cortex, parietal cortex, frontal cortex, occipital cortex, perirhinal cortex, and temporal cortex of AD brain. The association between augmented expression of GMF and APs/NFTs in AD was also evaluated. Double immunofluorescence labeling for GMF and APs show strongly GMF‐positive cells with neuron and glia morphology are co‐localized with APs and NFTs. The astrocytes adjacent to the APs display ramified processes and express GMF and GFAP, indicative of an activated phenotype associated with inflammation. Our results provide strong evidence that under conditions of neuro degeneration in AD, the expression of GMF is significantly upregulated. These results imply that augmented expression of GMF is associated with AD pathology.(Supported by VA Merit Review award and NIH/NINDS grant NS‐47145)
Role of 4‐hydroxynonenal in oxidative stress induced signaling
Authors: Chaudhary Pankaj , Sharma Rajendra , Sharma Abha , Vatsyayan Rit , Yadav Sushma , Hadley Marcus , Awasthi Sanjay , Awasthi Yogesh Chandra 
Published: June 2021

ObjectivesWe have shown that 4‐HNE induces signaling for apoptosis in cells via death receptor (Fas) mediated extrinsic and p53‐mediated intrinsic pathways. However, the nature of interactions of 4‐HNE with the components of these signaling pathways are not understood. During present studies, we have elucidated the pathways of 4‐HNE induced signaling for apoptosis in HepG2 cells.ResultsOur results showed that 4‐HNE promoted apoptosis in HepG2 cells through activation of p53, Bax, p21, JNK, and caspase3. Our results also showed that exposure of HepG2 cells to 4‐HNE leads to the activation of Fas, Daxx, ASK1, JNK, and caspase3 and these effects of 4‐HNE could be inhibited by the over expression of hGSTA4‐4. 4‐HNE also induced translocation of Daxx from nucleus to cytoplasm which caused inhibition of apoptosis and the activation and the translocation of HSF1 to nucleus resulting in the up‐regulation of HSP70. We also demonstrate that 4‐HNE does indeed bind to Fas, Daxx, and p53 to affect Fas and p53 mediated signaling.ConclusionThese findings suggest that 4‐HNE generated during oxidative stress promotes apoptosis through multiple pathways, but upon sustained oxidative stress conditions, 4‐HNE may provide protection to cells by up regulation of heat shock proteins and by Daxx mediated inhibition of apoptosis.( NIH grants ES 012171, EY04396 (YCA) and CA 077495 (SA))
Hyperactive mice show elevated D2High receptors, a model for schizophrenia: calcium/calmodulin‐dependent kinase II alpha knockouts
Authors: Novak Gabriela , Seeman Philip 
Published: June 2021

The cerebral frontal cortex of schizophrenia patients shows elevated levels of RNA for calcium/calmodulin‐dependent protein kinase II beta (CaMKIIβ). In addition, recent research shows that animal models for schizophrenia consistently show elevated levels of dopamine 2 receptors in their high‐affinity state (D2High), the major target for antipsychotic medication. The present study was done to examine whether an alteration in the levels of CaMKIIβ, in respect to CaMKIIα, could lead to altered levels of D2High receptors.We used quantitative real‐time PCR to measure CaMKIIα and CaMKIIβ mRNA levels in heterozygous and wild type CaMKIIα knock‐out mice, which show reduced CaMKIIα levels, and a D2R binding assay to determine the levels of D2High.The heterozygous CaMKIIα knock‐out mice, which show features analogous to schizophrenia, showed a 50% reduction in CaMKIIα mRNA, and no change in CaMKIIβ in the frontal cortex, as expected, but also a 29% upregulation of CaMKIIβ mRNA in the striatum and a 2.8‐fold increase in D2High receptors. In addition, we found that the CaMKII inhibitor, KN‐93, markedly reduced D2High states in rat striatum.The elevated levels of CaMKIIβ mRNA in the striatum suggest that this enzyme may increase D2High in animals and possibly in schizophrenia itself.
Validity of Maternal Assessment of Infant Breastfeeding Behavior: A Cross‐cultural Comparison
Authors: Matias Susana L , Nommsen‐Rivers Laurie A , Chantry Caroline J , Cohen Roberta J , Dewey Kathryn G 
Published: June 2021

We evaluated the validity of two potential ‘screening’ questions asked of primiparous mothers about a breastfeeding episode on days 0 and 3 post‐partum (pp) in Peru (n=154) and the US (n=248). A dichotomous measure (suboptimal vs. optimal infant breastfeeding behavior) based on Infant Breastfeeding Assessment Tool scores recorded by trained observers was the reference. Sensitivity (Se) and Specificity (Sp) of maternal assessment of suckling behavior (suckled well continuously/intermittently vs. otherwise; MASB) and maternal satisfaction (very satisfied/satisfied vs. otherwise; MAS) with a breastfeeding episode were calculated for independent and parallel use. Depending on the day pp, Se of MASB was 36–63% in Peru and 44–57% in the US; Sp was 97% in Peru and 97–98% in the US. Se of MAS was 61–85% in Peru and 48–58% in the US; Sp was 64–73% in Peru and 89–92% in the US. Parallel use of both questions improved Se in the US (55–73%). Increasing the cut‐off of MASB improved Se on both days in both samples (85–99%), but reduced Sp (31–69%). The use of these screening questions may help identify breastfeeding dyads who would benefit from further lactation support, especially in settings where availability of specialized lactation evaluation is limited.Supported by grant R40MC04294 from the Department of Health and Human Services, Maternal Child Health Branch and by the Gifford Center for Population Studies at UC Davis.
Activation of mice skeletal muscle 6‐phosphofructo‐1 kinase by serotonin is mediated by PLC‐ β PKC dependent pathway
Authors: Coelho Wagner Santos , Sola‐Penna Mauro 
Published: June 2021

Serotonin (5‐HT) is implicated in regulation of many physiological and pathological events, presenting the ability to stimulate glucose uptake on skeletal muscle. We previously reported that 5‐HT activates 6‐phosphofructo‐1‐kinase (PFK) from mice skeletal muscle in a non insulin‐dependent pathway, through 5‐HT2A receptor subtype. This study aims to gain further insights into the mechanisms involved in glucose metabolism control by 5‐HT, through enzyme activity assays and western blotting analysis using mice muscle stimulated with 5‐HT and inhibitors of signaling cascade intermediates. Here we show that activation of PFK promoted by 5‐HT is prevented by the PLC‐β inhibitor U‐73122, with concomitant reduction in 5‐HT‐mediated tyrosine phosphorylation of PFK. Moreover, 5‐HT effects are mimicked by phorbol myristate acetate (PMA) and blocked by bisindolylmaleimide‐1 (BIS‐1), which respectively activates and inhibits protein kinase C (PKC). KN‐62, a calmodulin kinase II (CaMKII) inhibitor, abolishes serotonin‐induced PFK activation. Furthermore, 5‐HT promoted co‐immunoprecipitation of PFK and CaMKII, suggesting a modulation mechanism. Yet, 5‐HT fails to promote extracellular regulated kinase 1/2 (ERK 1/2) phosphorylation. Altogether, our results support evidences that glycolytic flux control promoted by 5‐HT occurs in a PLC‐β PKC dependent pathway.Research Support: CNPq, FAPERJ, FAF/FECD
Neurosteroid binding domains of the NMDA receptor NR2B and NR2D subunits
Authors: Cameron Krasi , Borkowski Brian , Kim Joon , Rhoads Sarah , Cassada Gray , Rocha Monica , Gentile Lisa 
Published: June 2021

AMPA, NMDA and kainate receptors belong to the ionotropic glutamate receptor (iGluR) family. As binding to glutamate, a major fast excitatory neurotransmitter, causes activation of these channels, they play an important role in synaptic plasticity, memory and learning. Our research focuses on understanding how these receptors are regulated, for potential applications in conditions such as Alzheimer's and Parkinson's disease.The data presented here is aimed at understanding the differential regulation of NMDA receptors by the endogenous neurosteroids pregnenolone sulfate (PS) and 3‐alpha‐hydroxy‐5‐beta‐pregnan‐20‐one sulfate (PREGAS). PS potentiates the activity of NMDA receptors containing an NR2B subunit while it inhibits those containing an NR2D subunit. PREGAS negatively regulates all iGluRs. Intrinsic and extrinsic fluorescence studies will be presented that confirm the binding of the NMDA NR2B S1S2 and amino terminal domains (ATD) to both PS and PREGAS. Unlike the NR2B subunit, the NR2D S1S2 domain does not bind to PS and PREGAS, however the NR2D ATD does bind to both neurosteroids. Data from isothermal titration calorimetry and Stern‐Volmer analysis will be presented to help differentiate the binding site of each of these neurosteroids on both the NMDA NR2B and NR2D subunits.
Impact of vitamin B‐6 availability on gluconeogenesis from amino acid precursors in human hepatoma (HepG2) cells
Authors: da Silva Vanessa Rodrigues , Ralat Maria , Gregory Jesse F. 
Published: June 2021

Endogenous glucose synthesis is crucial to the maintenance of adequate glucose supplies for the body. The conversions of glycine to serine and of serine and alanine to pyruvate are pyridoxal 5′‐phosphate (PLP)‐dependent, leading to a metabolic intermediate which can be used for gluconeogenesis. This study aims to assess the impact of vitamin B‐6 deficiency on gluconeogenesis from amino acid precursors in a human hepatoma cell line. HepG2 cells were cultured in vitamin B‐6 replete (2000 nM pyridoxal) or vitamin B‐6‐deficient Dulbecco's minimal essential medium containing 10, 20, or 50 nM pyridoxal. At steady‐state, intracellular PLP levels were 29.1, 37.6, 42.5, and 57.2 pmol/mg protein, respectively. Cells were cultured for 24h in media enriched with a [U‐13C3]serine tracer, and enrichment of intra‐ and extracellular serine, glycine, and glucose measured by GC/MS. Maximum glucose enrichment occurred as the singly‐labeled species at 4h. Investigation is underway evaluate the effect of vitamin B‐6 status on gluconeogenic rate.Grant Funding Source: UFL
Selenoprotein P reduces lipid hydroperoxides in human embryonic kidney cells following exposure to 15‐ hydroperoxyeicosatetraenoic acid
Authors: Rock Colleen , Moos Philip J 
Published: June 2021

Reactive intermediates formed during cell membrane metabolism can contribute to carcinogenesis through oxidative damage to DNA. Potential chemopreventive effects of selenium supplementation have been attributed to the antioxidant activity of selenocysteine residues in selenoproteins. Selenoprotein P (SelP) is an extracellular glycoprotein that functions both in selenium distribution and as an antioxidant protein. The major objective of this study was to determine the effect of SelP on lipid hydroperoxides produced during the metabolism of arachidonic acid by 15‐lipoxygenase‐1. Enzymatic reduction of 15‐ hydroperoxyeicosatetraenoic acid (15‐HpETE) by SelP was observed in a NADPH‐coupled biochemical assay. Diphenylpyrenylphosphin was used to measure lipid hydroperoxides in SelP treated human embryonic kidney (HEK‐293) cells following exposure to 15‐HpETE. Lipid hydroperoxides increased with 15‐HpETE treatment of cells, and SelP reduced this effect, even when the protein and metabolite were added simultaneously. This shows that SelP can work as an antioxidant enzyme against reactive intermediates formed during inflammation. This effect may serve to protect cells against the oxidative damage induced by these lipid metabolites.This work is supported by NCI grant CA115616