bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Cinema To Teach Symptoms of and Empathy for Patients with Brain Disorders
Authors: Mize Richard Ranney
Published: June 2021

Clinical videos which demonstrate deficits produced by CNS diseases are commonly used in health professional neuroscience courses. Although useful, they often show compliant patients who do not reveal feelings, and sometimes do not effectively demonstrate the deficit. We have begun using Hollywood movies to more dramatically illustrate the emotional as well as physical impact of neurological disorders upon the patient and family. Commercial DVDs that we use include: "Coming Home" (Metro‐Goldwyn Meyer, 1978) which shows the environment of a rehabilitation ward and the progression of a veteran as he struggles to live with his war‐induced paralysis; "Hilary and Jackie" (Universal Studios, 1999) which documents the loss of somatomotor skills of the classical cellist, Jacqueline DuPre, as she succumbs to multiple sclerosis; "Iris" (Buena Vista, 2001) which illustrates the devastation of Alzheimer's on the mind of the British poet Iris Murdoch and the effect of the disease upon her husband and family; and "Sylvia" (BBC, 2003) which dramatically evokes the quiet descent of the poet Sylvia Plath towards suicide after a life‐long battle with major depression. Student evaluations suggest that these cinematic experiences: 1) teach empathy for the patient's condition; 2) dramatically demonstrate the full constellation of symptoms; 3) connect the physical symptoms with the emotional experience of the disease process.
Interrelations between C4‐ketogenesis and C5‐ketogenesis in the perfused rat liver
Authors: DENG Shuang , Zhang Guofang , Kasumov Takhar , David France , Roe Charles R. , Brunengraber Henri
Published: June 2021

C5‐Ketone bodies, 3‐hydroxypentanoate and 3‐ketopentanoate, are products of odd‐chain fatty acid oxidation in liver. They are formed when patients with long‐chain fatty acid oxidation disorders are treated with anaplerotic triheptanoin (glycerol‐triheptanoate; Roe et al J. Clin. Invest. 110: 259, 2002). C5‐ketogenesis results in the transfer of part of the anaplerotic propionyl moiety of triheptanoin to peripheral tissues. We investigated interrelations between C4‐ and C5‐ketogenesis in rat livers perfused with heptanoate ± octanoate or octanoate ± heptanoate or propionate with different labeling patterns. While uptakes of octanoate and heptanoate were similar, the yield of C5‐ketone bodies from heptanoate was lower than that of C4‐ketone bodies from octanoate. This results from the diversion of propionyl‐CoA moiety of heptanoate to anaplerosis and gluconeogenesis. The competition between octanoate and heptanoate uptakes favors octanoate uptake over heptanoate uptake and C4‐ketogenesis over C5‐ketogenesis. C4‐ketogenesis and C5‐ketogenesis share the same acetyl‐CoA pool. Very little propionate is converted to C5‐ketone bodies because the kinetic properties of 3‐ketoacyl‐CoA thiolase do not allow the formation of 3‐ketopentanoyl‐CoA from propionyl‐CoA + acetyl‐CoA. Thus C5‐ketone body formation requires an odd‐chain fatty acid with at least 5 C. Supported by NIH.
Single‐cell Molecular Profiling of Adipogenesis on an Integrated CARS‐Confocal Raman Platform
Authors: Le Thuc L , Slipchenko Mikhail , Cheng Ji‐Xin
Published: June 2021

A single‐cell approach is employed to examine phenotypic variability during the transformation of 3T3‐L1 cells into fat cells. Using a coherent anti‐Stokes Raman scattering (CARS) microscope, adipogenic gene expression, insulin signaling, and glucose import are visualized simultaneously with lipid synthesis within a single cell. Whereas lipid synthesis is not correlated with expression of adipogenic genes, a strong dependence on insulin sensitivity is observed. Faster insulin degradation rate renders advanced‐stage cells more sensitive to further insulin stimulation than early‐stage cells. Consequently, increased kinase activity and glucose import in advanced‐stage cells are observed. Single‐cell profiling reveals insulin sensitivity, as determined by insulin degradation rate, is the origin of cell‐to‐cell phenotypic variability in drug‐inducible adipogenesis. Furthermore, CARS microscopy has been integrated with confocal Raman microspectroscopy for molecular profiling in single lipid bodies, which allowed us to distinguish de novo synthesis and fatty acid uptake pathways in lipogenesis.
Organelle‐targeting, red‐emitting fluorescent probes with superior photo‐physical properties for multiplexing with green fluorescent protein (GFP) in live cells
Authors: Coleman Jack , Xiang Yuejun , Shen Dee , Pande Praveen , Schwartz Geoffrey , Gattica Divina , Patton Wayne
Published: June 2021

Cell‐based assays are increasingly gaining in popularity in the biotechnology and pharmaceutical industry due to their high physiological relevance. As trends advance toward analysis of living biological systems, fluorescent proteins (such as GFPs) have become indispensable tools for live‐cell analysis. One important application of GFPs is fluorescence co‐localization imaging, a powerful approach for exploring the targeting of molecules to intracellular compartments and for screening of their associations and interactions. To date, however, concerted efforts in developing fluorescent organelle‐targeting probes, specifically tailored to working in concert with GFPs, has been limited. Photo‐conversion of red‐fluorescent dyes to fluoresce green and metachromatic artifacts wherein fluorescent dyes emit both red and green lead to spurious results in GFP co‐localization experiments. Additionally, many organelle‐targeting probes photo‐bleach rapidly, are subject to quenching upon concentration in organelles or only transiently associate with the target organelle, requiring imaging within a minute of dye addition. Consequently, a new family of red‐emitting, cell‐permeable probes that spontaneously localize to specific subcellular organelles was developed. It is demonstrated that the probes can be readily used in combination with other common UV‐ and visible‐light excitable organic fluorescent dyes and various fluorescent proteins in multi‐color imaging and detection applications. It is further shown that the probes emit in the Texas Red region of the visible spectrum, and are highly resistant to photo‐bleaching, concentration quenching and photo‐conversion.
Role of endocytosis on human metapneumovirus entry
Authors: Chang Andres , Schowalter Rachel M. , Dutch Rebecca Ellis
Published: June 2021

Human metapneumovirus (HMPV) is a significant cause of respiratory infections in children, the elderly, and immunocompromised individuals. Our lab recently demonstrated that HMPV differs from other members of the paramyxoviridae family in that a low pH stimulated HMPV F protein‐mediated cell‐cell fusion. This suggested a role of endocytosis as a mechanism of entry for this virus. In this study, we examined the effects of inhibitors of endosomal acidification or endocytosis on entry of a recombinant GFP‐expressing HMPV. Chemicals that increase endosomal pH resulted in a 30‐50% decrease in infection whereas infection in the presence of the dynamin inhibitor dynasore was decreased by up to 90%. Furthermore, HMPV infection was not affected by the presence of several protease inhibitors including E64D, suggesting that the low pH requirement for HMPV entry is not due to the need of low pH‐stimulated endosomal/lysosomal proteases. This suggests that, unlike what has been hypothesized for most paramyxoviruses, HMPV utilizes endocytosis for entry. This research was supported by research grant 1R21AI074783‐01 from the National Institutes of Health.
Trafficking of IgG by FcRn in Epithelial Polarized Cells
Authors: Massol Ramiro Horacio , Lencer Wayne
Published: June 2021

FcRn transports IgG and IgG‐opsonized antigens across polarized epithelial cells that line mucosal surfaces. IgG transport by FcRn is essential for IgG homeostasis, short‐term passive immunity and contributes to epithelial maintenance, inflammatory responses and host defense at mucosal surfaces. In this work we characterized some aspects of the endocytosis and intracellular trafficking of FcRn in epithelial polarized cells. We found that FcRn‐IgG complexes are internalized from either the apical or basolateral surface mainly via clathrin‐mediated endocytosis. These complexes are then transported into the recycling endosome (RE) after which a fraction of IgG is recycled back to the surface of origin or transcytosed to the opposite cell surface. A very small fraction of FcRn‐IgG complexes is delivered to late endosomes/lysosomes. Whereas the actin motor myosin Vb and the GTPase Rab25 regulate sorting of these complexes out of the RE specific for transcytosis, Rab11a regulates recycling to the basolateral membrane only. Finally, we found that IgG‐opsonized particles crosslink and divert FcRn from the typical transcytotic and recycling pathways into the degradative pathway. This unusual mode of transport of FcRn‐IgG‐particles to the lysosomes may be important for the initiation of antigen processing or for the neutralization of pathogenic microbes.
Dermacentor variabalis: Tick saliva inhibits osteosarcoma (Saos) cell migration and invasion
Authors: Poole Nina M , Kramer Carolyn , Coons Lewis B , Cole Judith A.
Published: June 2021

Previous studies from our laboratory show that tick saliva inhibits fibroblast migration in both wound healing and chemotaxis assays. These results lead us to examine the effect of tick saliva on the invasiveness of Saos cells. Assessment of cell migration using a blind well assay showed that saliva decreased basal cell migration by 57.78+5.17% and inhibited stimulated migration by 67.41+8.87%. In addition[j1], saliva reduced SaOS invasiveness by 40% as measured by a matrigel invasion assay. Since extracellular signal‐regulated kinase (Erk) is involved in cellular movement and aberrant Akt activation contributes to increased cell invasiveness, we measured the effects of tick saliva on Akt and ERK activation by EGF. While EGF stimulation of ERK was not affected by tick saliva, Akt activation [j2]by 1 ng/ml EGF was inhibited ~55% (from 2.21+0.37 to 1.22+0.32). Our data indicate that a constituent(s) in tick saliva inhibits the invasiveness of an osteosarcoma cell and suggest that suppression of Akt signaling may be involved. Since metastasis of primary tumors often occurs in cancer, our data suggest the possibility of therapeutic agent(s) in tick saliva.
Phosphorylation of Yeast Phosphatidate Phosphatase by Protein Kinase C
Authors: Su Wen‐Min , Han Gil‐Soo , Carman George M.
Published: June 2021

The yeast PAH1‐encoded phosphatidate (PA) phosphatase catalyzes the Mg2+‐dependent dephosphorylation of PA to yield diacylglycerol and Pi. This enzyme plays a major role in the regulation of lipid metabolism. PA phosphatase is a phosphoprotein in vivo, and contains putative phosphorylation sites for multiple protein kinases. In this study, we examined the phosphorylation of the enzyme by protein kinase C using recombinant PA phosphatase expressed in E. coli. Purified His6‐tagged PA phosphatase was active, and was phosphorylated by protein kinase C in a time‐ and dose‐dependent manner. In addition, protein kinase C activity was dependent on the concentrations of PA phosphatase (28 μg/ml) and ATP (4.2 μM). Phosphoamino acid analysis indicated that phosphorylation by protein kinase C occurs primarily at the serine residue. Mass spectrometry of phosphorylated enzyme identified Ser773 and Ser788 as phosphorylation sites. However, phosphopeptide mapping analysis indicated additional sites of phosphorylation. Phosphorylation analysis of truncated forms of PA phosphatase indicated that the additional phosphorylation sites were located at the C‐terminal and central regions of the protein. Supported by NIH grant GM 50679.
Probing the Interactions of Nanoparticles with Biological Systems
Authors: Chan Warren C. W.
Published: June 2021

Nanoparticles have many applications in biomedical imaging, clinical diagnostics and therapeutics. Strategies that can reproducibly prepare colloidal nanoparticles of a wide range of geometries with a tight size distribution have been achieved and unique size and shape dependent optical, magnetic, electrical, and biological properties have been discovered. In spite of what has been achieved so far, a complete understanding of the interactions of cells with nanoparticles at the molecular level is lacking. This has led to the inability to rationally design nanoparticles for biomedical applications or has led to the inability to establish a definitive conclusion on the toxicity of nanomaterials. This presentation will focus on recent findings of the size and shape dependent interactions of nanoparticles with receptors on the cell surface. Specifically, data pertaining to the effect of antibody‐coated gold and silver nanoparticle size on regulating membrane receptor internalization and their subsequent signaling will be discussed. The findings presented here may assist in the design of nanoscale delivery and therapeutic systems and provide insights into nanotoxicity.
A novel approach to bioremediation of chromium using bacterial strains resistant to oxidative stress
Authors: Frederick Tamlyn , Woodruff Peter
Published: June 2021

Bioremediation is a process by which microorganisms reduce or prevent the negative effects of pollutants and contaminants in the environment. It has emerged as an effective and relatively inexpensive method utilized to clean up toxic waste sites and polluted environments. Given its powerful applications, an important focus in this field is the genetic engineering of microorganisms to increase the rate of bioremediation. Many naturally occurring bacteria have been found to detoxify the environment by reducing Chromium (VI) to Chromium (III). Chromium (VI) is a carcinogen that enters the environment as a byproduct from factories and industrial waste sites. Reducing this contaminant from the hexavalent form to its trivalent form makes its less soluble in water and less likely to contaminate water supplies. A problematic byproduct of the reduction process is the generation of reactive oxygen species. Therefore, we hypothesize that protecting bacteria involved in bioremediation from this oxidative stress will improve their capacity for bioremediation. The aim of this research is to genetically engineer bacteria such as E. coli that have improved resistance to the oxidative stress associated with chromium (VI) reduction. These heightened defenses against oxidative stress should increase viability of bacteria and improve bioremediation of Chromium (VI).

This research was supported by a Faculty Research Award from Bowdoin College (PW) and a Howard Hughes Medical Institute fellowship (TF)
Significance of eFactor VIIIc in angiogenesis
Authors: Baksi Krishna , Saha Subiman , Martinez Juan A , Banerjee Aditi , Banerjee Dipak K
Published: June 2021

Factor VIIIc is a large regulatory cofactor glycoprotein in the blood coagulation containing 6 sequential domains arranged in the order of A1‐A2‐B‐A3‐C1‐C2. The circulating factor VIII protein however is a metal bridged heterodimer consisting of a heavy chain (A1‐A2‐B) and a light chain (A3‐C1‐C2). Its deficiency leads to a congenital bleeding disorder, Hemophilia A. We have shown expression of Factor VIIIc‐like glycoprotein in capillary endothelial cells (i.e., eFactor VIIIc) as a 270 kDa asparagine‐linked (N‐linked) glycoprotein in which the heavy chain Mr 210,000 and the light chain Mr 46,000 dalton are joined together by disulfide‐bridge(s). eFactor VIIIc expression preceding the endothelial cell proliferation and its N‐glycosylation upregulation by cAMP signaling correlating the cellular proliferation further supported its role in angiogenesis. The objective of our study has been to understand the role of eFactor VIIIc in the angiogenic process. The results supported that (i) inhibition of eFactor VIIIc N‐glycosylation with tunicamycin inhibited angiogenesis; (ii) cells cultured in the presence of anti‐Factor VIIIc monoclonal antibody failed to invade the Matrigel(tm) matrix; and (iii) tunicamycin treatment exhibited considerable inhibition of Matrigel(tm) invasion. Thus, we conclude that eFactor VIIIc is involved in matrix dissolution during tumor invasion of blood vessels by activating the matrix metalloproteinases (MMPs). Supported in part by grants G12‐RR03035 (KB) and the Susan G. Komen for Cure BCTR0600582 (DKB).
Validating the Golgi two‐hybrid assay's utility in studying glycosylated proteins
Authors: Shew Matthew A. , Dube Danielle H.
Published: June 2021

Despite its utility in detecting interactions among nuclear and cytosolic proteins, the traditional yeast two‐hybrid assay (Y2H) systematically misses the interactions between secreted and cell surface proteins that traffic through the secretory pathway en route to the outside of the cell. To overcome this limitation, the Kohler lab developed the Golgi two‐hybrid assay (G2H) which tests secretome protein‐protein interactions in the Golgi, where proteins have their full complement of post‐translational modifications. In the G2H, protein‐protein interactions are linked to a selectable parameter within the Golgi ‐ Och1 mannosyltransferase activity. Previous results by Dube and Kohler demonstrate that the G2H can successfully detect the known protein‐protein interaction between MyoD and Id2. Here we set out to validate the G2H's ability to detect the known glycosylation‐dependent interaction between polypeptide N‐acetylgalactosaminyltransferase‐2 (ppGalNAcT‐2) and its substrate Muc5AC. This work was supported by INBRE and the Camille and Henry Dreyfus Foundation.
Identifying amino acid residues in the yeast high mobility group protein HMO2 that are important for DNA binding
Authors: Rahman Wedad , Ray Sreerupa , Grove Anne
Published: June 2021

High mobility group (HMGB) proteins are non‐histone nuclear proteins that play a role in DNA repair. The functional roles of one of the HMG domains of the yeast homolog HMO2 were examined by identifying amino acid residues that are involved in DNA intercalation. Through site directed mutagenesis, a valine in the box A domain, predicted to intercalate between DNA base pairs, was substituted with alanine. A binding assay with linear DNA showed that the mutated protein has lower binding affinity compared to wild type HMO2, while both mutant and wild type proteins have similar affinity for supercoiled DNA. The ability of the mutant HMO2 to protect free DNA ends from exonuclease digestion was determined; both wild‐type protein and the mutated protein were able to protect the DNA from exonuclease activity, suggesting that the substitution does not affect the preferred binding to DNA ends characteristic of HMO2. DNA binding assays further showed that both wild‐type and mutant protein bound with only modest preference to DNA with one loop (tandem mismatches), but both bound with significant preference to DNA with two loops and to DNA with one loop and one abasic site. Evidently, the valine in HMO2 box A is important for 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.
Differential Biochemical Activities of MutSα and MutSβ in mismatch Recognition and Nucleotide Binding and Hydrolysis
Authors: TIAN LEI , Gu Liya , Li Guo‐min
Published: June 2021

MutSα (MSH2‐MSH6) and MutS ? (MSH2‐MSH3) are eukaryotic mismatch recognition proteins that are preferentially responsible for processing base‐base and small insertion/deletion (ID) mismatches in vivo, respectively, despite the fact that cells express 10‐fold more MutSα than MutS ?. To understand the mechanism underlying the differential mismatch recognitions by these two proteins, purified human MutSα and MutS ? were analyzed individually and competitively for their abilities to interact with a T‐G and an ID substrates. We show that MutSα has a KD of 26.5 nM and 38.2 nM for the G‐T and ID substrates, respectively, and MutS ? has a KD of 76.5 nM and 23.5 nM for G‐T and ID, respectively. Consistent with these results, competitive binding assays revealed the following relative binding affinities: MutS ?‐ID > MutSα‐T‐G > MutSα‐ID > MutS ?‐T‐G. Interestingly, MutS ?‐ID interaction can be greatly stimulated when the MutSα:MutS ? ratio is = 10, justifying why cells maintain a high MutSα:MutS ? ratio. Distinct ATP binding and hydrolysis activities of MutSα and MutS ? were observed. For MutSα, the MSH6 subunit preferentially binds ATP, but the MSH2 subunit preferentially binds ADP. In the case of MutS ?, both the MSH3 and MSH2 subunits interact with ATP and ADP, despite the fact that MSH3 has a higher affinity for ATP than MSH2 and that MSH2 possesses a higher affinity for ADP than MSH3. We also find that MutSα ATPase activity can be stimulated by DNA substrates, but DNA inhibits MutS ? ATPase activity. These distinct biochemical activities of MutSα and MutSβ may contribute to their preferential recognition of base‐base mismatches and ID mispairs, respectively.
Structural characterization of the extracellular domain of alpha‐neurexin:insights into synapse assembly
Authors: Miller Meghan , Comoletti Davide , Wilson Jennifer , Nakagawa Teru , Taylor Palmer
Published: June 2021

Neurexins are a family of pre‐synaptic cell‐adhesion membrane proteins that are important for synapse maintenance through contact with the postsynaptic protein, neuroligin. Several studies indicate that mutations in these genes may play an important role in autism and mental retardation. Each neurexin gene contains two promoters generating α‐ and β‐neurexins. Whereas the extracellular domain of β‐neurexin contains only a single LNS domain, α‐neurexin consists of six LNS domains, including the β‐neurexin domain, separated by three EGF domains. We have used single particle EM and small angle x‐ray scattering to obtain low‐resolution images of the extracellular domain of α‐neurexin, which offer insights into the macromolecular structure. Furthermore, epitope tagging by antibodies reveal the N‐ and C‐terminus. Using rigid body modeling and ab‐initio restoration approach, we have determined a structural model of the entire extracellular domain of α‐neurexin. The crystal structures of the individual LNS domains in neurexin and the β‐neurexin‐neuroligin complex supplement the analysis. Together with analytical ultracentrifugation, mass spectrometry, and gel filtration, we now have an understanding of the basic biophysical properties of α‐neurexin. These results show that α‐neurexin exists as a glycosylated monomer, with an extended conformation and some inherent flexibility between the LNS domains.
Neuroregulatory factors in traumatic brain injury
Authors: Kane Michael Jason , Kalmbach Keri H , Butler Carrie L , Dennis John S , Citron Bruce A
Published: June 2021

The mechanisms underlying neuron loss in central nervous system disorders are incompletely understood. We have previously identified dysregulation in several pathways important to neuronal health. In this study, we focus on identifying regulatory factors that could be used to protect and preserve neuronal function following traumatic brain injury (TBI). Elucidation of factors involved in neuronal death will provide targets to achieve therapeutic benefits during a narrow window of opportunity after TBI. Using an in vitro cell injury model, we identified several transcription factors that are upregulated that can result in a proapoptotic gene expression profile, including E26 avian leukemia oncogene 1 (ETS1), interferon regulatory factors (IRF3 and 6), MAD homolog 3 (Smad3), and Wilms tumor homolog (WT1). The expression levels of these factors were elevated about 1.7 fold and 3.5 fold in the case of ETS1. Our data indicate that inflammatory responsive transcription factors, implicated in apoptosis in other tissues, are also important in cell loss in the brain. The identification of these and other factors that regulate neuronal survival following TBI may represent an attractive target for pharmacological intervention. This study was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, the Bay Pines Foundation, and the Alzheimer's Association.
Notch/RBP‐J signaling is necessary for astrogliogenesis and the expression of astrogliogenic genes NFIA and Sox9
Authors: Janssens Derek , Johnson William , Sarah Sarala , Van derKlay Tiffany , Taylor Merritt
Published: June 2021

As neural stem cells (NSC) divide they can give rise to three classes of cells: neurons, glia and more stem cells (a process called self renewal). During development NSCs first primarily give rise to neurons (neurogenesis) and later give rise to glia (gliogenesis). In early development the Notch/RBP‐J signaling pathway allows NSCs to self renew by suppressing neuronal differentiation. As development progresses, Notch/RBP‐J signaling regulates the differentiation of NSCs into glia. Although it is known that Notch regulates self renewal through the expression of the Hes genes, the mechanism for Notch‐dependent gliogenesis has not been characterized.

We are determining if Notch signaling controls the expression of the astrogliogenic genes NFIA and Sox9. We have identified putative binding sites for RBP‐J, a transcriptional co‐regulator of canonical Notch signaling, in the promoter regions of both genes. Using quantitative PCR and immunofluorescence techniques we found that disrupting Notch signaling in neural progenitors of the developing CNS by deletion of the gene for RBP‐J (called Nestin Cre Rbpsuh fl/fl mice) causes a significant decrease in both NFIA and Sox9 expression. This demonstrates that Notch/RBP‐J signaling is necessary for the expression of these astrogliogenic genes, and suggests that Notch employs different signaling pathways for NSC self‐renewal and for gliogenesis.
Molecular Dynamic Simulation of Human mu‐Opioid Receptor
Authors: Lindner Marc I , Anderle Gloria T , Calcagnetti Daniel
Published: June 2021

The ?‐opioid receptor is a transmembrane G‐protein coupled receptor that binds opiates and plays a role in analgesia, physical dependence, respiratory depression, and euphoria. Recently, a class of drugs has been developed to treat overdose. These drugs bind more strongly to the receptor than the opiate, acting as inhibitors. The antagonist?β‐Funaltrexamine (β‐FNA), binds irreversibly to the receptor through a covalent bond. It has been speculated that β‐FNA binds in a two‐step process. Our goal was to use the method of molecular dynamic simulation to verify the binding behavior of β‐FNA. The human ?‐opioid receptor has not been crystallized; however, it is a member of the rhodopsin family of proteins. Since the sequence of the human ?‐opioid receptor is known, a homology model was built based on the bovine rhodopsin crystallography structure. A second model based on the rat ?‐opioid receptor homology model of Mosberg, was also generated. Both models were simulated with β‐FNA in the reputed binding site using the Amber suite of molecular dynamic programs under NPT and periodic boundary conditions at 310 K in explicit TIP3 water. The resulting structures were then compared.
Biochemical and structural characterization of innate immunity kinase complex proteins
Authors: Call R. Jason , Ghirlando Rodolfo , Bell Jessica K.
Published: June 2021

Protein‐protein interactions form the communication link from membrane bound receptors to their downstream cellular targets.

Objective

Our long‐term goal is to examine the protein‐protein interactions responsible for the activation and regulation of the Toll‐like receptor 3 (TLR3) signaling cascade that leads to type I interferon production. In the current study we have targeted the kinase scaffold proteins, TRAF family member‐associated NF‐κB activator (TANK) and NAK‐associated protein 1 (NAP1).

Methods

Bacterial expression systems have been used to produce recombinant target protein and refolding methods developed for insoluble targets. Initial biochemical characterization of recombinant targets was completed using circular dichroism (CD), gel filtration (GF) and analytical ultracentrifugation (AUC).

Results

The protein domain comprising the N‐terminal region of NAP1 can be successfully refolded as assessed by CD, while TANK is soluble. Both constructs form homo‐oligomers as determined by GF. AUC experiments show that the NAP1 N‐term behaves as an elongated dimer in vitro. Gel filtration studies of TANK's kinase interaction site show that it elutes as a dimer species.

Conclusions

The scaffold proteins, TANK and NAP1, form stable homo‐oligomers via their N‐terminal regions. Funding from the Am. Cancer Society (RJC) and supported in part by the NIH intramural research program, NIDDK (RG).
Regulation of NF‐kB Nuclear Translocation by AKIP and PKAc
Authors: King Charles C , Sastri Mira , Taylor Susan S.
Published: June 2021

Regulation of NF‐?B Nuclear Translocation by AKIP and PKAcA Kinase‐Interacting Protein/Breast Cancer Associated Gene 3 (AKIP/BCA3), a protein with three splice variants that is up‐regulated in breast cancers, binds the N‐terminus of the catalytic subunit of PKA (PKAc), and results in nuclear translocation of PKAc. Recent data has also demonstrated that AKIP/BCA3 binds NF‐?B and regulates transcription, suggesting that AKIP/BCA3 functions as a molecular shuttle for NF‐?B and a direct regulator of transcription. PKAc phosphorylates NF‐?B at serine 276, and can enhance nuclear translocation. Using a biochemical approach, the rate of GFP‐p65 nuclear translocation was monitored with different stimuli in the presence or absence of AKIP or a peptide that disrupts the interaction of AKIP and PKA. Enhanced nuclear translocation of p65 was observed in the presence of over‐expressed AKIP with TNFα and this could be blocked by the disrupting peptide. Addition of the cAMP analog (8‐CPT‐cAMP) was able to modestly induce p65 nuclear translocation in the absence of TNFα, and the combination of TNFα and 8‐CPT‐cAMP had a decreased rate of p65 nuclear translocation compared with TNFα alone. Taken together, these results suggest that AKIP regulates the rate of translocation of p65 into the nucleus in a PKA‐dependent manner.
FRET studies on distinct cAMP/PKA signaling induced by beta adrenergic receptor subtypes
Authors: Xiang Yang
Published: June 2021

cAMP/PKA activation represents a key signaling mechanism for neurohormonal stimulation of diversified physiological processes. Using real‐time, FRET‐based imaging of PKA activity in neonatal cardiac myocytes, we report that sustained activation of PKA induced by β adrenoceptors (βAR) dictates signaling propagation for substrate phosphorylation and myocyte contraction. Activation of βARs in wild‐type myocytes induces strong and sustained PKA activities, which are rapidly attenuated upon washing away agonist or adding antagonist to the cells. The sustained PKA activities promote signaling propagation to the sarcoplasmic reticulum for phosphorylation of phospholamban and increases in myocyte contraction rate. Addition of antagonist after βAR stimulation significantly blocks PKA phosphorylation of phospholamban, and rapidly reduces contraction rate increases. Moreover, Stimulation of β1AR subtype induces PKA activities similar to those in wild‐type cells. In contrast, stimulation of β2AR subtype induces strong initial activation of PKA similar to those induced by β1AR; however the activities are rapidly decreased to baseline levels. The transient PKA activities are sufficient for phosphorylation of the activated β2ARs, but not phospholamban. Further analysis reveals that phosphosdiesterase 4 is the major enzyme family that shapes PKA activities under βAR stimulation. Inhibition of phosphosdiesterase 4 extends β2AR‐induced PKA activities, promotes PKA phosphorylation of phospholamban, and ultimately enhances myocyte contraction responses. Together, our data has revealed insights into kinetics of PKA activities in signaling propagation under neurohormonal stimulation.
Molecular characterization of the EMR2 receptor signaling pathways in human neutrophils
Authors: Chen Zih‐Ying
Published: June 2021

The EGF‐TM7 receptors belong to a subfamily of adhesion‐GPCRs that are predominantly expressed on myeloid leukocytes. We have recently shown that ligation of EMR2, one of the EGF‐TM7 members, by 2A1 mAb primes numerous neutrophil functions, including cell adhesion, migration, release of reactive oxygen species and degranulation (FASEB J. 22(3):741‐751, 2008). However, the precise signaling mechanism mediated by EMR2 remains elusive. Herein, we examined the activation status and kinetics of signalling molecules well known in neutrophil activation. Our results show rapid increased phosphorylation of mitogen‐activated protein kinases (MAPK), p38, ERK, and JNK, in neutrophils upon EMR2 ligation. In addition, ligation of EMR2 by 2A1 was shown to reduce the LPS‐mediated inhibitory effect on spontaneous neutrophil apoptosis. Taken together, our results suggested that the function of EMR2 receptor in neutrophil is most likely signaled via MAPKs. (Research support: CMRPD330083)
Association of UDP‐Glucuronosyltransferases with 14‐3‐3 Chaperone Protein and Kinase‐dependent Signaling Partners
Authors: Basu Nikhil K , Basu Mousumi , Mitra Partha S , Banerjee Rajat , Owens Ida S
Published: June 2021

UDP‐glucuronosyltransferase (UGT) isozymes eliminates from the body a vast number of lipophilic metabolites, therapeutic drugs and chemical toxins that include dietary constituents and environmental contaminants by increasing water solubility via conversion to glucuronides. Recently, we uncovered the fact that UGT isozymes require phosphorylation for activity. Phosphorylation, regulated via signaling, occurs on serine/threonine and tyrosine residues. Inasmuch as UGT1A7His and PKCε co‐localized in 1A7‐transfected COS‐1 cells, we attempted to co‐immunoprecipitate the two proteins with anti‐His from solubilized cellular extracts and analyze for related proteins. Curcumin (75 µM) caused complete disassociation of β‐COP and PKCε and dephosphorylation of UGT1A7, whereas 500 nM calphostin‐C completely disassociated PKCε and β‐COP. The coordinate loss of PKCε and β‐COP with dephosphorylation of UGT following curcumin or calphostin‐C treatment indicates these factors are likely located in a complex that supports UGT1A7 activity. Similarly, both PKCα and PKCδ, but not PKCε, co‐localized and co‐immunoprecipitated with UGT1A10‐His. Further, finding 1A7 in a 225‐kDa complex with phospho‐serine 14‐3‐3 chaperone protein during purification demonstrated UGT is associated within a signaling unit consistent with regulated phosphorylation, predictably maintaining stability of UGT proteins. ε
Significance of activation loop phosphorylation in protein kinase A studied by H/D exchange and X‐ray crystallography
Authors: Steichen Jon Martin , Kuchinskas Michael P , Iyer Ganesh H , Li Sheng , Woods Virgil L , Taylor Susan S
Published: June 2021

Protein phosphorylation is possibly the most important mechanism for regulation in eukaryotic cells. Over 500 protein kinases are encoded by the human genome and the catalytic subunit of cAMP‐dependent protein kinase (PKA) serves as a prototype for the entire family of protein kinases. The activation loop is a critical motif for all protein kinases and in many kinases, such as PKA, the activation loop must be phosphorylated in order for the kinase to be active. The inactive form of PKA can be purified by mutating an arginine to alanine (R194A) in the activation loop which prevents autophosphorylation of T197, the critical phosphorylation site. The unphosphorylated enzyme is less active and less stable based on unfolding and kinetic studies. Hydrogen/deuterium exchange coupled to mass spectrometry was used to compare the inactive and active states of PKA. Much of the enzyme is more solvent exposed in the inactive state including regions distant from the activation loop, demonstrating that phosphorylation induces large allosteric changes in the enzyme. Additionally, diffracting crystals of the R194A mutant have been grown and these crystals will provide a structural comparison of the phosphorylated and unphosphorylated states of PKA. This research was supported by NIH Grant GM19301.
Altered retinoic acid (RA)‐induced lineage specification of mouse embryonic stem cells (mESCs) following a transient pulse of a histone deacetylase inhibitor.
Authors: Fritsch Michael Kevin , Alexanian Ruben , Singer Don B. , Murdoch Fern E.
Published: June 2021

RA treatment during mESC differentiation results in predominantly neural specification. A transient pulse of the histone deacetylase inhibitor, Trichostatin A (TSA), during early mESC differentiation via embryoid body (EB) formation results in maintenance of undifferentiated characteristics. Our objective was to determine whether a pulse of TSA followed by RA alters neural lineage specification. Undifferentiated mESCs are exposed to 100 nM of TSA during the first 24h of 3 days of EB formation. EBs are plated and RA added for an additional 4 days. Selected RT‐PCR shows repressed neural marker expression and only a small increase in a few endoderm markers. Gene expression array studies analyzed using a lineage annotated gene database demonstrates a marked increase in expression of numerous placental genes and confirms diminished neural gene expression. Immunofluorescence for the trophoblast marker Plac1, shows increased levels in TSA‐pulsed, RA‐treated cells with minimal expression in RA‐only differentiated cells. Conversely, the neural specific product, Doublecortin, is highly expressed in the RA‐only treated cells and shows diminished protein levels in TSA‐pulsed, RA‐treated cells. We conclude that transient TSA treatment prior to RA induces a dramatic lineage switch characterized by repressed neural differentiation and marked trophoblast induction. Supported by NIH grant RO1DK064243 to MKF.
Effects of Ziram on Human Natural Killer Cells MAP kinases p38 and p44/42
Authors: Taylor Thyneice Rochelle , Whalen Margaret M
Published: June 2021

Human natural killer cells (NK) are lymphocytes that can recognize and kill virally infected cells, antibody coated cells, and tumor cells without any prior sensitization. Ziram is used as a fungicide in agriculture and as an accelerating agent in the production of latex. Previous studies have shown that ziram inhibits the ability human NK cells to lyse tumor target cells. Mitogen activated proteins kinases (MAPK) are important signaling enzymes in NK cells. Exposure of NK cells to another environmental contaminant, which also blocks NK lytic function, tributyltin (TBT), induced phosphorylation (activation) of MAPK. Thus, disturbances in MAPK activation state could contribute to TBT (and possibly ziram) ‐induced loss of lytic function. The current study examines whether ziram causes MAPK activation. The MAPKs, p44/42, p38 and JNK were examined. A 10 minute exposure to 10 µM or 5 µM ziram caused a significant increases in phosphorylation of p44/42 (3 fold and 2 fold, respectively). A 10 minute exposure to 10 µM, 5 µM, or 2.5 µM ziram caused a significant increase in phosphorylation of p38 (4 fold, 3 fold, and 2 fold, respectively. These results suggest that MAPK activation may be part of the mechanism by which ziram inhibits NK lytic function, and that MAPK activation may be an effect that is shared by several environmental contaminants that have been shown to decrease NK lytic function.

Supported by NIH grant S06 GM008092‐33.
The function of MAP kinase phosphatase‐1 in Gram‐negative sepsis
Authors: Wang Xianxi , Frazier Joshua W. , Meng Xiaomei , Nelin Leif D. , Liu Yusen
Published: June 2021

MAP kinase phosphatase (Mkp)‐1 acts as a feedback control mechanism that attenuates p38 and JNK in innate immune cells exposed to Toll‐like receptor (TLR) ligands. We have previously demonstrated that mice deficient in Mkp‐1 gene are highly sensitive to bacterial products including lipopolysaccharide (LPS) and more readily succumb to shock and multi‐organ failure. However, the role of Mkp‐1 in the immune responses to actual Gram‐negative bacteria remains unclear. We investigated the role of Mkp‐1 during Gram‐negative sepsis using Mkp‐1 knockout mice and E. coli as a model system. Compared to wild type mice, Mkp‐1 knockout exhibited earlier and greater mortality after infected intravenously with live E. coli. Histological analysis revealed more severe pulmonary edema and markedly hemoconcentration in the knockout mice than in the wild type mice. Interestingly, plasma from the infected wild type mice, but not those from the infected Mkp‐1 knockout mice, exhibited hyperlipidemia. Surprisingly, the Mkp‐1 knockout mice had a substantially greater bacterial burden than did the wild type mice in both the blood and spleens of the knockout mice. Gentamicin prevented mortality in wild type mice but had no effect on Mkp‐1 knockout mice. Our results indicate that Mkp‐1 is a crucial immune regulator determining the prognosis after infection and plays an important role in host defense far beyond simply regulating cytokine production.
Cloning and expression of parasitic mitogen‐activated protein kinase
Authors: Apablaza Pablo , Shah Swati , Patel Samta , Sierkierka John , Dyer James
Published: June 2021

Mitogen‐activated protein kinases (MAPKs) are involved in a wide variety of processes in many types of cells and in many different organisms. Human MAPKs are known to be responsive to stress stimuli and are involved in apopotosis and cell differentiation. Parasite and plant MAPKs are also involved in stress responses. These MAPKs have been identified in the Apicomplexan parasite Toxoplasma gondii and also in the malarial parasite Plasmodium falciparum. The purpose of this research is to clone and express these MAPKs in a heterologous expression system so that futher biochemical characterization of the protein may be done.
Angiotensin II Stimulated Angiogenesis is Dependent on p38 MAPK and is Inhibited by Statins
Authors: Naggar Jack Clement , Zhang Yali , Park Ho‐Jin , Georgescu Serban P , Galper Jonas B
Published: June 2021

Recent data suggest that angiogenesis plays a role in the development of atherosclerotic plaques. Previous studies have demonstrated that Angiotensin II (AII) induces angiogenesis in various systems, but its mechanism is not well understood. Our study tries to determine the molecular mechanism of the AII stimulated angiogenesis and the relationship between lipid metabolism and AII signaling. Here we show that the HMG‐CoA reductase inhibitor, Simvastatin, interferes with AII stimulated angiogenesis via the inhibition of p38 MAPK activation. Western blots using extracts of human umbilical cord vein endothelial cells (HUVEC) treated with AII demonstrated a time‐dependent increase in the phosphorylation of p38 (2.8±0.3 fold at 10 min and 6.0±0.2 fold at 20 min). Pretreatment of HUVEC with Simvastatin dose‐dependently inhibited AII stimulated p38 phosphorylation (62±19% reduction at 0.01 ?M and 83±17% reduction at 1 ?M). AII stimulated angiogenesis by 1.6±0.3 fold in a HUVEC Matrigel tube formation assay. Simvastatin treatment showed a dose‐dependent inhibition of AII stimulated tube formation (60% inhibition at 0.1 ?M and complete inhibition at 1 ?M). p38 inhibitor, SB203580, inhibited AII stimulated tube formation 98±18%. Our results suggest that AII induces angiogenesis, at least in part, via activation of p38, and that statins interfere with AII stimulated angiogenesis via the inhibition of this pathway.
Effect of aqueous Methanolic extract of Senna alata and Senna podocarpa on some hematological parameters in rats
Authors: Adebesin Olumide Adedapo , Okpuzor Joy
Published: June 2021

Senna alata and Senna podocarpa are widely used herbals by the southwestern populace in Nigeria to treat various kinds of diseases, so the long term (63 days) effect of the leaf aqueous Methanolic extract of these plants on some hematological parameters were investigated in albino rats given a daily dose of 1ml by gavage, of 0‐, 200‐, 600‐ and 1000‐mg/kg body weight of extract, to assess its safety. At the termination of the experiment, blood was collected via ocular puncture and the effect of the extract on packed cell volume (PCV), white blood cell (WBC), platelets and the differential white blood cells count were evaluated. S.alata at 200‐, 600‐, 1000‐ and S.podocarpa at 200‐ mg/kg body weight significantly increased (P=0.003; 0.001; 0.004 and 0.01 respectively) the PCV levels over that of the control. The results suggests that these extracts may contain substance(s) that improves erythropoiesis and thus reduce anemia in rats.
Evaluation of flavonoids as agents to inhibit breast cancer
Authors: Yadegarynia Sina , Bremer Martina , White J Brandon
Published: June 2021

Flavonoids (flavones and isoflavones) are abundant in plants and are known to be associated with the reduced risk of many chronic diseases. We screened 8 flavonoids for their ability to induce cell death in 4 breast cancer cell lines. These cell lines were selected based upon different phenotypes for signaling pathways to elucidate if certain pathways were required. Apigenin, chrysin, daidzein, genistein, kaempferol, luteolin, naringenin, and quercetin were screened at different concentrations and incubation times in MDAMB231, SKBr3, MCF7, BT‐474, and ZR75 cells. CellTiter‐Glo and trypan blue exclusion assays were performed at least three times to measure cell viability. Statistical data analysis was conducted for CellTiter‐using a four‐factor ANOVA model for drug, cell line, drug concentration, and time. F‐tests followed by Tukey's multiple comparison procedure were used to find statistically significant differences in cell death. All flavonoids were capable of inducing cell death. The strongest cell death occurred in all cell lines with apigenin or luteolin. Kaempferol and chrysin induced cell death but only at the highest concentrations used. Daidzein, genestein, naringenin, and quercetin killed cell lines very weakly. Our results suggest only certain flavonoids are capable of significant cell death across a variety of breast cancer cell lines. Support of research was through NSF‐REU Program at SJSU.
Meta‐analysis of the General Effects of a Standardized Lycium barbarum Fruit Juice Shown in Randomized, Double‐blind, Placebo‐controlled Human Clinical Studies
Authors: Amagase Harunobu , Hsu Chiu‐Hsieh Paul
Published: June 2021

Four randomized, double‐blind, placebo‐controlled clinical trials with a total of 161 participants (18‐72 years age) were pooled to study the general effects of orally consumed 120 ml/day of Lycium barbarum, as a standardized juice, GoChi. A questionnaire consisting of clinical symptoms graded 0‐5 and the score changes were analyzed between GoChi and placebo at pre‐ and post‐intervention in a significance level of 5%. Meta‐analysis by R program was conducted for score changes using standardized mean differences (treatment effect sizes) and the associated standard errors. Under a random effects model, GoChi group (n=81) had statistically significant improvements in weakness, stress, mental acuity, ease of awakening, shortness of breath, focus on activity, sleep quality, daydreaming and overall feelings of health and well‐being compared to the placebo group (n=80). Under a fixed effects model, GoChi group had additional statistically significant improvements in fatigue, depression, circulation and calmness compared to the placebo. For binary outcomes, odds ratios (OR) and the standard errors were used in the Mantel‐Haenszel method. GoChi group had statistically significant higher chance of improvements indicated by OR in fatigue, dizziness and sleep quality compared to the placebo. Three studies had statistically significant heterogeneity in procrastination and shoulder stiffness.