Dissertations / Theses on the topic 'Biochemistry and Cell Biology N.E.C'
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1
Buchanan, Fritz G. "Endogenous Alkylglycerol Functions As a Mediator of Protein Kinase C Activity and Cell Proliferation." Digital Commons @ East Tennessee State University, 1997. https://dc.etsu.edu/etd/2885.
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To explore the possibility that 1-O-alkyl-sn-glycerol (alkylglycerol) may serve a regulatory role in the control of cell proliferation or PKC activity, we examined the ability of alkylglycerol to influence PKC activity and subcellular distribution as well as the ability of alkylglycerol to effect cell proliferation. MDCK cells grown to confluence show a loss of PKC activity associated with the membrane, as reported in fibroblasts. Preconfluent cultures of MDCK cells have a high level of PKC activity associated with the membrane. However, treatment of preconfluent cultures with alkylglycerol causes a reduction of PKC activity. A similar inhibition was observed with alkylglycerol when cells were treated with TPA, an activator of PKC. To confirm that alkylglycerol was exerting an effect directly on PKC, alkylglycerol was shown to inhibit PKC activity in vitro in a dose dependent manner. Since PKC exists as a family of closely related isozymes, we have determined the effects of growth arrest and alkylglycerol treatment on PKC $\rm\alpha,\ \epsilon,\ and\ \zeta$ (expressed in MDCK cells). The active forms of PKC $\alpha$ and $\epsilon$ are lost early in the growth of MDCK cells during the endogenous accumulation of alkylglycerol and synthetic alkylglycerol inhibits the membrane form of PKC $\alpha$ and $\epsilon.$ However, alkylglycerol inhibits the TPA induced translocation of PKC $\alpha$ but not $\epsilon$ suggesting a differential inhibition among these isoforms. Neither TPA or alkylglycerol had any effects on the distribution of PKC $\zeta.$ To examine the effect of alkylglycerol on cell proliferation, Swiss 3T3 cells were used. GLC analysis shows that 3T3 cells accumulate alkylglycerol in a similar manner as MDCK cells. Since this accumulation occurs just prior to cell growth arrest, the effects of alkylglycerol on preconfluent cells was observed. Preconfluent cultures of 3T3 cells were treated with alkylglycerol on day 1 of growth. After 8 days of culture, the treated group showed a slower growth rate and saturation density. Furthermore, after these cells were reseeded in the absence of alkylglycerol, the original growth rate and saturation density returned. Thus alkylglycerol induces a decrease in cell proliferation without causing any detrimental effects. Similarly, alkylglycerol was found to inhibit the induction of mitogenesis by TPA (a PKC dependent pathway) and these effects were shown not to be stereospecific. To further investigate the effect of alkylglycerol on cell proliferation, the content of the monoglycerides in ras-transformed cells was analyzed. These cells have lost contact dependent growth arrest indicating a disruption of cell growth regulation. We observed a massive increase in the content of alkylglycerol during the culture of ras transformed cells. This increase is 3 fold higher than MDCK or 3T3 cells. This raises the possibility that alkylglycerol may be the end result of an increased number of cell-cell contacts. We have observed an increase in the accumulation of alkylglycerol in normal and ras-transformed cells. This accumulation is accompanied by a decrease in PKC activity and alkylglycerol was shown to be a potent in vitro inhibitor of PKC. Similarly, alkylglycerol was shown to inhibit PKC $\alpha$ under stimulation by TPA. Alkylgylcerol is a inhibitor of the TPA induced induction of mitogenesis and slows the growth rate of proliferating cultures of 3T3 cells. These results indicate that the endogenous ether-linked glycerolipid, alkylglycerol, is a regulator of cell proliferation through its inhibitory effects on protein kinase C. (Abstract shortened by UMI.)
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Williams, Kendra Allana. "Phosphorylation of Histone Deacetylase 6 within its C-terminal Region by Extracellular Signal Regulated Kinase 1." Scholar Commons, 2013. http://scholarcommons.usf.edu/etd/4792.
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Gu, Cong. "Superoxide Dismutase C Modulates Macropinocytosis and Phagocytosis in Dictyostelium Discoideum." FIU Digital Commons, 2018. https://digitalcommons.fiu.edu/etd/3887.
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Macropinocytosis and phagocytosis, two actin-dependent and clathrin independent events of endocytosis, enable the cells such as macrophages and neutrophils to either internalize pathogens and initiates the human innate immune response or serve as a direct entry route for productive infection of pathogen. Dictyostelium discoideum, soil-living amoeba, a unicellular eukaryote that could professionally internalize fluid phase or particles several folds more than that of macrophages and neutrophils. Additionally, multiple key signaling pathways are conserved between Dictyostelium and mammalian cells, including pathways affecting small GTPases Ras and Rac and their downstream effectors, and F-Actin remodeling. All these traits makes Dictyostelium an excellent model organism to study the process pf macropinocytosis and phagocytosis.
Upon internalization of the prey, these macropinocytes and phagocytes are often in an environment of increased production of superoxide radicals in the prey-containing vesicles, which helps stimulates the downstream signaling pathways to digest the prey inside. However, the mechanism of how superoxide regulates the process of macropinocytosis and phagocytosis is not fully understood. We had previously reported that Dictyostelium cells lacking Superoxide dismutase C (SodC) exhibited aberrantly high level of active RasG, high basal level of Phosphatidylinositol-3,4,5-triphosphate (PIP3), and severe chemotaxis defects. Now we report that sodC- cells displayed aberrant endosomal vesicle trafficking, significantly compromised particle uptake and defective cell to substratum matrix adhesion compared to that of wild type cells. By using high resolution live imaging microscope we also show that sodC- cells have defects in F-Actin remodeling at the phagocytic rim extension and F-Actin depolymerization of the nascent phagosome. Interestingly, the introduction of overexpressing of cytoplasmic superoxide dismutase (SodA), redox insensitive RasG (C118A) or treatment of PI3K inhibitor LY294002 in sodC- cells significantly rescued the defects of endosomal vesicle trafficking, particle uptake and adhesion. This project suggests that superoxide dismutase C regulates the endosomal vesicle trafficking, phagocytosis and cell to substratum matrix adhesion through the RasG/PI3K signaling axis in Dictyostelium cells.
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Unsworth, Amanda J. "The role of protein kinase C in platelet activation." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:114582b8-185a-41f5-958c-77038fb185df.
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The Protein kinase C (PKC) superfamily is a key regulator in platelet activation with individual isoforms playing distinct roles. This thesis focuses on the role of the novel PKC isoforms downstream of several agonists using both pharmacological and genetic approaches and human and mouse platelets. Quantification of the protein levels of PKC isoforms identified different levels of the five major PKC isoforms expressed in human platelets and also differences between levels of the same isoform in human and mouse platelets. Use of a selection of broad spectrum and isoform-specific inhibitors, identified both positive and negative novel roles for PKC in the regulation of human and mouse platelets. A net positive role for PKC was found in GPVI, Clec-2, and PAR receptor signalling, with classical isoforms of PKC playing a major role in aggregation and dense granule secretion. A novel negative regulatory role was also identified in the regulation of ADP-induced platelet activation for PKC~, and both PKCE and PKC~ in human and mouse platelets respectively. Gene knock-out mouse models confirmed a positive regulatory role for PKCe in allb~3 outside-in signalling but identified no other regulatory role for PKCe in agonist induced platelet activation. Despite this relatively minor role, functional redundancy was identified between PKCe and PKCE isoforms in haemostasis, as tail bleeding was significantly increased in mice deficient in both novel isoforms. The work presented here identifies key roles for the PKC superfamily in the complex regulation of platelet activation, with different isoforms supporting and limiting the process of thrombus formation and haemostasis.
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Hiyama, Jun. "Isolation and characterisation of N-glycans of ovine and human luteinizing hormones." Thesis, University of Auckland, 1991. http://hdl.handle.net/2292/1989.
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Gonadotrophic hormones are heterodimeric glycoproteins and their N-glycans attached to specific amino acid residues are currently thought to play important roles in hormonal biosynthesis, secretion and function. The studies reported in this thesis aimed at isolation and characterisation of structural properties of the N-glycans on ovine and human luteinizing hormones. Initially, chromatographic methods were developed using reverse-phase HPLC for the analytical separation of the three human pituitary glycoprotein hormones and their subunits. Separation of intact oLH and its subunits was also effected by a single HPLC step. A preparative procedure was developed for the efficient purification of hLH and hTSH from crude human pituitary extracts using hydrophobic chromatography which gave highly purified hormones in good yields and with high biological activities. This method did not significantly influence the hormones' extensive charge heterogeneity and it offered potential advantages in the characterisation of their carbohydrate structures. A preparative scheme was developed for the isolation of the N-linked oligosaccharides from each glycosylation site of o- and hLH. Charge heterogeneity of oligosaccharides, which were released by hydrazinolysis from subunits and glycopeptides, was characterised by anion-exchange HPLC. 1H-NMR analysis showed that the structures of all three N-glycans on hLH were highly heterogeneous but mainly diantennary complex-type, with site-specific patterns of terminal sialylation and sulphation as well as core-fucosylation. Sulphated/sialylated and/or disialylated oligosaccharides were the major components at each site. A set of new mono- and disialylated oligosaccharides with the terminal sequence NeuAcα2-6GalNAcβ1-4G1-cNAcβ1-2Manα1-3 was identified. This finding suggested unique site-specific terminal sialylation of oligosaccharides at Asn 78 (hLHα) by an unknown α2-6 sialyltransferase(s) in the human pituitary gonadotroph cell. Each glycosylation site in oLH had a distinct set of oligosaccharides ranging from mainly monosulphated hybrid-type at the two sites of oLHα to predominantly disulphated diantennary complex-type on oLHβ. Core-fucosylation also differed at each site. These results suggested that processing of the oligosaccharides of the α- and β-subunits by α-mannosidase II and α1-6 fucosyltransferase was differently regulated by protein structure in oLH. Whereas hCG, hLH and oLH share similar biological activities, no apparent relationship between their N-glycan structures was found, which suggested that specific branching and peripheral structures of N-glycans on LH and hCG may not be essential for biological function, although the N-glycan nearer the N-terminus of the α-subunit of hCG has been implicated in hormonal activity.
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Lumngwena, Evelyn Ngwa. "The impact of HIV-1 subtype C Envelope N-glycosylation on DC-SIGN meditated modulation of DC function to facilitate transmission or enhance viral pathogenesis." Doctoral thesis, University of Cape Town, 2017. http://hdl.handle.net/11427/27096.
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N-glycosylation plays an important role in Envelope (Env) function and may be involved in the modulation of the immune response to HIV-1 infection. In this study, we hypothesized that Env N-glycosylation may affect viral pathogenesis by influencing Env structure and function. Furthermore, we also postulated that differences in Env glycosylation could affect interactions between Env and DC-SIGN of dendritic cells (DCs), activating alternative signalling pathways which stimulate the release of different immune modulators. We generated pseudovirus of eighteen Env clones (PSVs) with variable number and position of potential N-glycan sites (PNGs) and compared their ability to infect TZM-bl cells, bind to Raji+ DC-SIGN cells, trans-infect TZM-bl cells when captured by either Raji-DC-SIGN cells or monocyte-derived dendritic cells (MDDCs) and modulate MDDC signaling by investigating the release of Interleukin-10 (IL-10) and other immune modulatory cytokines and MAPK activation. Entry efficiency, DC-SIGN binding and trans-infection varied widely across all clones. The level of IL-10 secreted by MDDCs in response to PSV stimulation varied 32-fold. The induction of IL-10 secretion by purified gp140 confirmed that Env was the viral component that stimulated the secretion of IL-10 via interaction with DC-SIGN and potentially other undefined receptors. PSV and purified gp140 stimulated MDDC signaling via ERK and JNK phosphorylation, while p38 was not activated. The addition of recombinant DC-SIGN lowered the levels of secreted IL-10 and ERK /JNK phosphorylation, suggesting that DC-SIGN plays a role in these responses. As Env mannosylation correlated with DC-SIGN binding, five highly conserved Env PNGs (241, 262, 386, 392, and 448) previously identified to carry high mannose type N-glycans and hence thought to be involved in DC-SIGN binding were deleted in two Env clones by site-directed mutagenesis to confirm their importance in Env function. The potential role of these PNGs in Env entry efficiency, DC-SIGN binding, trans-infection, induction of MDDC IL-10 secretion and activation of MAPK phosphorylation was determined. Deletion of these sites significantly affected the entry efficiency, DC-SIGN binding, trans-infection and MDDC IL-10 secretion, with one Env clone proving to be more sensitive to mutation than the other. This suggests that PNGs influence Env function in a clone-specific manner. As deletion of highly conserved PNGs abrogated Env function we used sequence analysis to identify PNGs involved in binding DC-SIGN and inducing MDDC IL-10 secretion. We grouped PSVs based on the presence or absence of specific PNGs in Env sequences and compared entry efficiency, DC-SIGN binding, trans-infection, stimulation of MDDC IL-10 secretion and induction of MAPK phosphorylation. Three Env PNGs were significantly associated with entry efficiency (N356, N392, and N674), and three sites (N289, N356 and 674) were significantly associated with trans-infection while N674 also influenced DCSIGN binding. The majority of MDDC donors secreted higher levels of IL-10 when stimulated with PSVs that carried PNGS at N130 (p = 0.0016) and N332 (p = 0.0039) and lacked N674 (p = 0.033). When Envs were graded on whether they had 0, 1, 2 or 3 of the PNGs (e.g. -130, -332, +674; -130, +332 and +674, etc.) those that carried either one of the PNGs or the entire induction motif (N130+ N332+ N674-) significantly stimulated MDDCs to secrete higher levels of IL-10 than those that completely lacked the motif (p = 0.0335 and p = 0.0304, respectively). As the presence of N674 was linked to reduction in all functions of Env, it is likely that the presence of an N-glycan at this site affected Env structure and could skew the analysis. Excluding N674 indicated that the presence of PNGs at position 130 and 332 was sufficient to induce significantly higher IL-10 release than those that had either none or one of these sites (p = 0.0053). When we determined whether N130 and N332 were enriched in subtype C acute infection Envs, these sequences were not enriched with PNGs at either N130 or N332 compared to chronic infection viruses. However, when IL-10 levels were compared between MDDC donors stimulated with PSV of either acute or chronic infection clones, those from early infection significantly enhanced MDDC secretion of IL-10 (p = 0.0039). This suggests that even though PNGs at 130 and N332 could be involved in inducing MDDC IL-10 secretion, it is not the only requirement for enhanced stimulation. Although Env differentially activated ERK and JNK phosphorylation, ERK phosphorylation did not correlate with IL-10 secretion, suggesting that this MAPK signaling pathway was not solely responsible for triggering the release of MDDC IL-10 and other regulatory cytokines. PSVs also stimulated the release of TNFα, IL-1β, IL-6, IL-8, MIP-1a, and MIP-1b while having no effect on IL-12 levels. This suggests that HIV-1 binding to DCs in the genital tract could change the dynamics of DC immune responses, deregulating their cytokines secretion and destabilising the Th0 cell differentiation to facilitate viral survival and thus productive clinical infection. We therefore conclude that HIV-1 variants differentially stimulate MDDCs to release immunosuppressive IL-10 and that transmitted founders could be better at modulating immune responses in the genital tract compared to chronic infection variants.
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Dagälv, Anders. "Role of Heparan Sulfate N-sulfation in Mouse Embryonic Development." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-123474.
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Heparan sulfate (HS) is a sulfated glycosaminoglycan expressed by all cells in the body. It is found at the cell surface and in the extracellular matrix where it binds a large amount of various ligands including growth factors and morphogens. HS is important for building up morphogen gradients during embryonic development and to act as coreceptors for signaling molecules. Many different Golgi enzymes are involved in the biosynthesis of HS. It is known that some of these enzymes interact with each other but not how the whole biosynthesis machinery works or how the cell regulates the structure of the HS that it produces. In this thesis, cells and mice deficient in two of these biosynthetic enzymes, glucosaminyl N-deacetylase/N-sulfotransferase-1 (NDST1) and the isoform NDST2 have been studied. NDSTs perform the first modifications during biosynthesis where they replace N-acetyl groups on N-acetyl-glucosamine units with sulfate groups. It is known that deficiency of NDST1 is lethal, while lack of NDST2 only results in abnormal connective tissue type mast cells. Here it is shown that deficiency of both NDST1 and NDST2 is embryonically lethal. The embryonic stem (ES) cells extracted from the inner cell mass of double knockout blastocysts show in addition an impaired differentiation capacity compared to wild-type ES cells and fail completely to differentiate into cardiac muscle cells which NDST1-/-, NDST2-/- and wild-type ES cells all do. Cultured mast cells that lack NDST2 produce heparin that is low-sulfated compared to wild-type HS. To our surprise, we could show that mast cells deficient in NDST1 instead produce a more highly sulfated heparin than wild-type cells. We use a model that predicts that the biosynthesis enzymes work together in a multienzyme complex, the GAGosome, to explain our results. We hypothesize that NDST1 has a higher affinity for the GAGosome than NDST2 which only in the absence of NDST1 gets incorporated into the enzyme complex. When all GAGosomes contain NDST2, a more highly sulfated glycosaminoglycan chain will be synthesized. A splice variant of NDST1, NDST1S, has also been studied. We could show that NDST1S lacks enzyme activity but that it probably has the capacity to incorporate into GAGosomes. Overexpression of NDST1S results in altered structure of the HS produced by the cells. We speculate that expression of the splice variant during development may be one way to regulate HS structure.
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de, Jesus Tristan J. "Novel Mechanisms of Immune Regulation by NF-kappaB c-Rel." Case Western Reserve University School of Graduate Studies / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1576176282590614.
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Giblin, Sean. "Investigating cell lineage specific biosynthesis of tenascin-C during inflammation." Thesis, University of Oxford, 2018. http://ora.ox.ac.uk/objects/uuid:8c7306d8-53cf-4131-a134-f74885e37cc9.
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The extracellular matrix (ECM) is a complex network of molecules secreted by cells, which is essential for providing structural support and facilitating cell processes including adhesion, migration and survival. Tenascin-C is an immunomodulatory ECM protein that exhibits limited expression in healthy tissues, but is transiently elevated at sites of tissue injury, and is persistently expressed in chronic inflammatory diseases and tumours. Alternative splicing of 9 of tenascin-C's fibronectin type III-like domains (FnIII- A1, A2, A3, A4, B, AD2, AD1, C and D) generates enormous diversity in form; yielding 511 possible isoforms. Post-transcriptional modification of tenascin-C has been studied in cancer and during development where disease and tissue specific isoforms exhibit distinct adhesive, migratory and proliferative effects. However, little is known of how tenascin-C is expressed or alternatively spliced during inflammation. This study characterises inflammation and disease specific tenascin-C isoforms made by immune cells and fibroblasts, and investigates their functional relevance. Biosynthesis and alternative splicing of tenascin-C was examined using standard curve qPCR, ELISA, Western blot and confocal immunocytochemistry in resting and activated primary human immune cells, dermal fibroblasts, and in synovial fibroblasts isolated from healthy controls and from osteoarthritis (OA) and rheumatoid arthritis (RA) patients. Based on these data, three recombinant proteins comprising FnIII domains AD2-AD1, B-C-D and B-AD2-AD1-C-D were cloned, expressed and purified, and their impact on cell behaviour including adhesion, morphology and migration was assessed. Basal tenascin-C expression was lower in myeloid and lymphoid cells than fibroblasts, and was induced in all following inflammatory stimulation. Tenascin-C expression was elevated in disease with RA and OA synovial fibroblasts containing higher levels than healthy controls. Alternative splicing following cell activation was cell-type specific: all FnIII except AD2 and AD1 were upregulated in dendritic cells and macrophages, in T-cells all FnIII remained unchanged with FnIII A1 absent; and no change in splicing was observed in activated dermal fibroblasts. Normal and OA synovial fibroblasts exhibited similar tenascin-C splicing patterns, but FnIII B and D were specifically elevated in RA. Functional analysis revealed differences in the adhesion, morphology and migration of myeloid cells and dermal fibroblasts cultured on FnIII AD2-AD1, B-C-D, B-AD2-AD1-C-D and full length tenascin-C substrates; FnIII B-C-D promoted MDDC migration while B-AD2-AD1-C-D promoted fibroblast adhesion, compared to full length tenascin-C. For the first time, this study reveals differences in tenascin-C biosynthesis and alternative splicing by immune cells and fibroblasts following activation with inflammatory stimuli; and starts to reveal how alternative splicing of tenascin-C may influence the behaviours of both stromal and immune cells types during inflammation and in inflammatory diseases.
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Smith, Abigail O. "Defining the Role of c-Jun N-terminal Kinase (JNK) Signaling in Autosomal Dominant Polycystic Kidney Disease." eScholarship@UMMS, 2021. https://escholarship.umassmed.edu/gsbs_diss/1141.
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Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people. It primarily is caused by mutations in the transmembrane proteins Polycystin-1 (PKD1) and Polycystin-2 (PKD2). The most proximal effects of polycystin mutations leading to cyst formation are not known, but pro-proliferative signaling must be involved for the tubule epithelial cells to increase in number over time. The stress-activated mitogen-activated protein kinase (MAPK) pathway c-Jun N-terminal kinase (JNK) promotes proliferation in specific contexts and is activated in acute and chronic kidney disease. Previous work found evidence of JNK activation in cystic tissues (Le et al., 2005) and others showed that JNK signaling is activated by aberrant expression of PKD1 and PKD2 in cell culture (Arnould et al., 1998; Arnould et al., 1999; Parnell et al., 2002; Yu et al., 2010) but the contribution of JNK signaling to cystic disease in vivo has not been investigated.
This body of work describes the use of conditional and germline deletion of Pkd2, Jnk1 and Jnk2 to model ADPKD and JNK signaling inhibition in juvenile and adult mice. Immunoblots and histological staining were used to measure JNK activation and evaluate the effect of JNK deletion on cystic disease. Results show that Pkd2 deletion activated JNK signaling in juvenile and adult mice. Reduction of JNK activity significantly reduced cystic burden in kidneys of juvenile Pkd2 mutant mice. This correlated with reduced tubule cell proliferation and reduced kidney fibrosis. The improvement in cystic phenotype was driven primarily by Jnk1 deletion rather than Jnk2. JNK signaling inhibition in adult Pkd2 mutants significantly reduced liver cysts when mice were aged six months. JNK inhibition reduces the severity of cystic disease caused by the loss of Pkd2 suggesting that the JNK pathway should be explored as a potential therapeutic target for ADPKD.
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Desai, Shraddha R. "Role of Protein Kinase C-iota in Glioblastoma." Scholar Commons, 2011. http://scholarcommons.usf.edu/etd/3070.
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The focus of this research was to investigate the role of protein kinase C-iota (PKC-é) in the regulation of Bad function, a pro-apoptotic member of the Bcl-2 family and Cdk7 function, a master cell cycle regulator in glioblastoma.
The results were obtained from the human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-é co-localized and directly associated with Bad as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-é directly phosphorylated Bad at phospho specific residues, S112, S136 and S155 which in turn induced inactivation of Bad and disruption of the Bad/Bcl-XL dimer. Knockdown of PKC-é by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-é may be a Bad kinase. Since, PKC-é is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-é/Bad pathway. Treatment with PI(3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-é activity and subsequent phosphorylation of Bad suggesting that PKC-é regulates the activity of Bad in a PI (3)-kinase dependent manner.
Robust expression of PKC-é is a hallmark of human glioma and benign and malignant meningiomas, however, little is understood about its role in glioma cell proliferation. The cyclin dependent kinase activating kinase complex (CAK), comprises of cyclin dependent kinase 7 (Cdk7), cyclin H and MAT1, is the master cell regulator. Cdk7 phosphorylates its downstream cyclin dependent kinases (cdks) and promotes cell proliferation. Results show that PKC-é directly associated and phosphorylated Cdk7 at T170. Furthermore, Cdk7 phosphorylated its downstream target, cyclin dependent kinase 2 (cdk2) at T160. Purified PKC-é was also observed to phosphorylate endogenous as well as exogenous Cdk7. PKC-é knockdown with siRNA, PDK1 siRNA and (PI) 3-kinase inhibitors, Wortmannin and LY294002 treatment exhibited corresponding reduction in phosphorylation of Cdk7 and subsequently cdk2. In addition, PKC-é knockdown reduced cell proliferation; led to cell cycle arrest and also induced apoptosis. Thus, these findings suggest the presence of a novel PI (3)-kinase/PKC-é/BAD mediated cell survival and PI (3)-kinase/PKC-é/Cdk7 mediated cell proliferation pathway.
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Pillai, Prajit P. "Role of Protein Kinase C-iota in Neuroblastoma and the Effect of ICA-1, a Novel Protein Kinase C-iota Inhibitor on the Proliferation and Apoptosis of Neuroblastoma Cells." Scholar Commons, 2011. http://scholarcommons.usf.edu/etd/3292.
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Protein Kinase C-iota (PKC-é), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-é confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of the dissertation was three-fold: First to study the role of PKC-é in the proliferation of neuroblastoma. Secondly, to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-é inhibitor in neuroblastoma cell proliferation and apoptosis. Finally, to analyze whether PKC-é could self-regulate its expression. Cyclin dependent kinase 7 (Cdk7) phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. Our data shows that PKC-é is an in-vitro Cdk7 kinase and that neuroblastoma cells proliferate via a PKC-é/Cdk7/cdk2 cell signaling pathway. ICA-1 specifically inhibits the activity of PKC-é but not that of PKC-zeta (PKC-æ), the closely related atypical PKC family member. The IC50 for the kinase activity assay was approximately 0.1µM which is 1000 times less than that of aurothiomalate, a known PKC-é inhibitor. The phosphorylation of Cdk7 by PKC-é was potently inhibited by ICA-1. ICA-1 mediates its antiproliferative effects on neuroblastoma cells by inhibiting the PKC-é/Cdk7/cdk2 signaling pathway. ICA-1 (0.1µM) inhibited the in-vitro proliferation of BE(2)-C neuroblastoma cells by 58% (P=0.01). Additionally, ICA-1 also induced apoptosis in neuroblastoma cells. Interestingly, ICA-1 did not affect the proliferation of normal neuronal cells suggesting its potential as chemotherapeutic with low toxicity. Hence, our results emphasize the potential of ICA-1 as a novel PKC-é inhibitor and chemotherapeutic agent for neuroblastoma.
Bcr-Abl has been shown to regulate the activation of the transcription factor ELK-1 which in turn regulates the expression of PKC-é. Alternatively, we hypothesize that PKC-é can self regulate its expression by indirectly regulating the activity of Elk-1 in an ERK1 dependent manner. Our preliminary data shows that there was robust increase in the expression as well as association of PKC-é and Elk-1 in actively proliferating neuroblastoma cells suggesting a potential role of PKC-é in regulating the activity of Elk-1. Analysis of the subcellular fractions also presented a similar increase in the association between PKC-é and Elk-1 in the nuclear fraction of actively proliferating cells as compared to cytoplasm. Interestingly, the nuclear expression of PKC-é was also found to be higher in these cells, suggesting that PKC-é translocated to the nucleus in actively proliferating cells and regulated the transcriptional activity of Elk-1. However, our data from in-vitro kinase activity demonstrated that PKC-é was not an Elk-1 kinase but that it increased the phosphorylation of Elk-1 in the presence of ERK1, an upstream kinase of Elk-1 in the Bcr-Abl mediated regulatory pathway of PKC-é. This suggested that ERK1 was integral to the self-regulatory activity of PKC-é. In conclusion, we hypothesize that the self-regulatory mechanism of PKC-é is initiated by the translocation PKC-é into the nucleus where it activates ERK1. This promotes the activation of its downstream target Elk-1 which subsequently upregulates the expression of PKC-é
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Johnston, Alyssa N. "A Ternary Drug Delivery Complex to Target CD44 Over Expressing Cancerous Cell Lines." Kent State University Honors College / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1335737666.
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Farley, Brian M. "Sequence and Target Specificity of the C. elegans Cell Fate Specification Factor POS-1: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/629.
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In most metazoans, early embryogenesis is controlled by the translational regulation of maternally supplied mRNA. Sequence-specific RNA-binding proteins play an important role in regulating early embryogenesis, yet their specificities and regulatory targets are largely unknown. To understand how these RNA-binding proteins select their targets, my research focused on the C. elegans CCCH-type tandem zinc finger protein POS-1. Embryos lacking maternally supplied POS-1 die prior to gastrulation, and exhibit defects in the specification of pharyngeal, intestinal, and germline precursor cells. To identify the regulatory targets that contribute to the POS-1 mutant phenotype, we set out to determine the sequence specificity of POS-1 in vitro, and then use this information to identify regulatory targets in vivo.
Using a candidate-based search, we identified a twelve-nucleotide fragment of the mex-3 3' untranslated region (3' UTR) to which POS-1 binds with high affinity. Using quantitative fluorescent electrophoretic mobility shift assays, I determined the affinity of the RNA-binding domain of POS-1 for a panel of single nucleotide mutations of this sequence, and then defined a consensus binding element based on this dataset. POS-1 recognizes the degenerate element UAU 2-3 RDN 1-3 G, where R is any purine (adenosine or guanine), and D is any base except cytosine. A bioinformatics analysis revealed the presence of this element in approximately 40% of C. elegans 3' UTRs, suggesting that POS-1 is capable of binding to and perhaps regulating many transcripts in vivo. POS-1 binding sites alone are not sufficient to pattern the expression of a reporter, suggesting that other factors may contribute to POS-1 specificity.
To address the mechanism of POS-1-mediated translational regulation, I investigated the translational regulation of the C. elegans Notch homolog glp-1. Previous work demonstrated that glp-1 translation is repressed in the early embryo in a POS-1-dependent fashion, though it was not clear if this regulation was direct. The glp-1 3' UTR contains two POS-1 binding sites within five nucleotides of each other, and these sites are within a thirty nucleotide region of the 3' UTR required for proper spatiotemporal translation of glp-1. The POS-1 sites overlap with a negative regulatory element that is recognized by GLD-1, and a positive regulatory element recognized by an unknown factor. Both POS-1 and GLD-1 bind to an RNA containing these sites in vitro, and POS-1 competes with GLD-1 for binding. Both proteins are required for translational repression of a glp-1 3' UTR reporter in embryos. Furthermore, only one of the two POS-1 binding sites is required for repression, and the required site is wholly contained within a previously characterized positive regulatory element. Based on this, we propose that POS-1 does not regulate its targets by recruiting regulatory machinery, but instead by competing with factors that do. Thus, sites of POS-1 regulation are highly context dependent, which may contribute to POS-1 specificity.
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Sutherland, Ashley B. "Fabrication of Responsive Polymer Brushes for Patterned Cell Growth and Detachment." Wright State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=wright1376981495.
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Anderson, Ryan L. "Fatty Acid Amides and Their Biosynthetic Enzymes Found in Insect Model Systems." Scholar Commons, 2018. https://scholarcommons.usf.edu/etd/7467.
Full textAbstract:
A fatty acid amide is precisely as the name suggests: A fatty acid (CHn-COOH), in which the hydroxyl group of the carboxylic acid is displaced by an amine functional group from a biogenic amine (R-NH2), ultimately forming an amide bond. Furthermore, these fatty acid amides can be composed of a variety of different acyl chain lengths donated by the fatty acid and a myriad of different biogenic amines. Thus, these molecules can be subdivided in a number of different ways including the separation of short chain (acetyl to heptanoyl) and long chain (palmitoyl to arachidonoyl) and also based off the biogenic amine type. The long chain fatty acid amides quickly gained the interest of the scientific community through the discovery of anandamide (N-arachidonoylethanolamide), which was found to be the endogenous ligand for the cannabinoid receptor-1 (CB1) found in the mammalian brain. This particular neural molecule is an N-acylethanolamide, which is one specific classification of long chain fatty acid amide. However, there exist other types of long chain fatty acid amides including the N-acylglycines, primary fatty acid amides (PFAMs) and N-acylarylalkylamides. Yet, despite the type of fatty acid amide, it has been shown many of these types of molecules are synthesized using a type of N-acyltransferase. These N-acyltransferases are believed to be members of the GCN5-related superfamily of N-acyltransferases (GNAT), which share the feature of being able to accept acyl-CoA thioester substrates. This dissertation will discuss and demonstrate the extraction of all types of the aforementioned classifications of long chain fatty acid amides but will have a particular focus on the N-acylarylalkylamides. Elucidating more about the biosynthetic pathways and metabolic routes of the long chain fatty acid amides could lead to the development of potential therapeutics and pest control agents. We have determined Drosophila melanogaster arylalkylamine N-acyltransferase like 2 is responsible for the in vivo biosynthesis of N-acyldopamines. We have also demonstrated Bombyx mori is another suitable model systems for the study of long chain fatty acid amides, as three insect arylalkylamine N-acyltrasnferase from Bombyx mori (Bm-iAANAT) were found to share some homology in primary sequence (25-29%) to AAANTL2 in Drosophila melanogaster. We show herein that one of these enzymes is able to catalyze the formation of long chain N-acylarylalkylamides in vivo. The change in the transcription of these enzymes was tracked to try and understand if these enzymes serve a focused purpose in the physiological development of the insect. If it is found one of these Bm-iAANAT are crucial for growth, it may elucidate a general function of the enzyme, which may be able to inhibit growth of specific insects that are known pests, while not targeting endangered insects like Apis melliferra (honey bee). Understanding this would help in the eventual creation of targeted insecticides on specific insect pests Furthermore, a novel panel of fatty acid amides was characterized and quantified in extracts from this organism via LC-QToF-MS, ultimately showing it is very possible the Bm-iAANATs are performing this catalysis in vivo.
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Barnard, Sandra H. "Amalgamation of Nucleosides and Amino Acids in Antibiotic Biosynthesis." UKnowledge, 2013. http://uknowledge.uky.edu/pharmacy_etds/20.
Full textAbstract:
The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading frame which encodes a putative serine hydroxymethyltransferase (SHMT). The revelation of this shared open reading frame resulted in the proposal that this putative SHMT catalyzes an aldol-type condensation reaction utilizing glycine and uridine-5’-aldehyde, resulting in the GlyU core. The enzyme LipK involved in A-90289 biosynthesis was used as a model to functionally assign this putative SHMT to reveal its functions as an l-threonine: uridine-5’-aldehyde transaldolases. Biochemical analysis indicates enzymatic activity is dependent upon pyridoxal-5’-phosphate, is non-reactive with alternative amino acids, and produces acetaldehyde as a co-product. Structural characterization of the enzymatic product is consistent with (5’S,6’S)-GlyU indicating that this enzyme orchestrates a C-C bond breaking and formation resulting in two new stereocenters to make a new l-a-amino acid. The same activity was demonstrated for the LipK homologues involved in the biosynthesis of muraminomicin, A-503083, and A-102395. This l-threonine: uridine-5’-aldehyde transaldolase was used with alternative aldehyde substrates to prepare unusual l-a-amino acids, suggesting the potential for exploiting this enzyme to make new compounds.
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Peters, Jeffery. "Effects of Nicotinamide Riboside and Beta-hydroxybutyrate on C. elegans Lifespan." Digital Commons @ East Tennessee State University, 2020. https://dc.etsu.edu/honors/531.
Full textAbstract:
The nicotinamide riboside (NR) form of vitamin B3and the ketone body ß-hydroxybutyrate (BHB) are two of the most promising natural compounds yet identified for the treatment of aging and aging-related diseases. Forms of vitamin B3are precursors for the synthesis of the coenzymes nicotinamide adenine dinucleotide (NAD(H)) and nicotinamide adenine dinucleotide phosphate (NADP(H)). In aged cells levels of NAD+decline, decreasing metabolism and decreasing activity of protective sirtuin protein deacetylases. In aged cells NR, but not more common forms of vitamin B3, boost NAD+levels. BHB is naturally produced by the body when individuals fast or consume a ketogenic (KD) or calorically restricted (CR) diet. These diets have been shown to extend lifespan in mice, while they are also protective in many disease models. Caenorhabditis elegans, a roundworm with a short mean lifespan of roughly 2 to 3 weeks depending upon the temperature, is used as a model system to study aging. BHB has been previously shown to increase lifespan by roughly 20% when administered to C. elegans.We administered NR and BHB individually and together to C. elegans starting at two different developmental stages (larval stages 1 and 4) and measured lifespan. We found that administration of 20 mM DL-BHB decreased lifespan when first given at the L1 stage, while it robustly increased lifespan when first given at the L4 stage. Administration of 0.5 mM NR increased lifespan when first given at L1, with only a very slight increase when first given at L4. When initiating administration at L1, NR greatly mitigated the BHB-mediated decline in longevity, however, NR did not increase BHB-mediated lifespan extension when first administered at L4.
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Dahl, Göran. "Kinetic studies of NS3 and NS5B from Hepatitis C virus : Implications and applications for drug discovery." Doctoral thesis, Uppsala universitet, Institutionen för biokemi och organisk kemi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-98868.
Full textAbstract:
The aim of these studies was to increase our understanding of the non-structural proteins 3 and 5B (NS3 and NS5B) from the hepatitis C virus (HCV), and thereby contribute to the development of new and better drugs against HCV. By studying NS3 with substitutions identified to be associated with resistance to NS3 inhibitors in clinical trials (R155Q, A156T and D168V) it was found that not all inhibitors were affected, indicating that cross-resistance can be avoided. Substitutions at position 526 and 528 in the helicase domain of this bifunctional enzyme were introduced and the effect on the protease was investigated. These substitutions affected protease inhibition, showing that the helicase can influence the protease. This interplay between the two domains is also involved in the discovered activation of the enzyme at low inhibitor concentrations. Being a case of "enzyme memory", the phenomenon stresses the importance of using full-length NS3 for enzymatic assays. Inhibitors with novel designs, with presumed increased stability in vivo, were developed and, even though they were found to be of low potency, provide alternative ideas of how to design an inhibitor. Detailed information about the interaction between NS3 and its protein cofactor NS4A or several protease inhibitors were determined using a direct binding assay. The rate constants of the inhibitor interactions were affected by NS4A and it was also possible to visualize time-dependent binding inhibitors. A good correlation between interaction data (Kd or koff) and inhibition data (Ki) or replicon data (EC50) was also seen. The same approach was used for studying the interactions between NS5B and several non-nucleoside inhibitors, providing information of the chemodynamics and giving insights into inhibitor design. Taken together, all these studies have resulted in new information about, and new tools with which to study, NS3 and NS5B. This is of great importance in the struggle to find new and potent drugs, leading to a cure for HCV infection.
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Wu, Sijia. "Novel Mechanisms Regulating Dopamine Transporter Endocytic Trafficking: Ack1-Controlled Endocytosis And Retromer-Mediated Recycling." eScholarship@UMMS, 2001. http://escholarship.umassmed.edu/gsbs_diss/887.
Full textAbstract:
Dopamine transporters (DAT) facilitate high-affinity presynaptic dopamine (DA) reuptake in the central nervous system, and are required to constrain extracellular DA levels and maintain presynaptic DAergic tone. DAT is the primary target for addictive and therapeutic psychostimulants, which require DAT binding to elicit reward. DAT availability at presynaptic terminals ensures its proper function, and is dynamically regulated by endocytic trafficking. My thesis research focused on two fundamental questions: 1) what are the molecular mechanisms that control DAT endocytosis? and 2) what are the mechanism(s) that govern DAT’s post-endocytic fate? Using pharmacological and genetic approaches, I discovered that a non-receptor tyrosine kinase, activated by cdc42 kinase 1 (Ack1), stabilizes DAT plasma membrane expression by negatively regulating DAT endocytosis. I found that stimulated DAT endocytosis absolutely requires Ack1 inactivation. Moreover, I was able to restore normal DAT endocytosis to a trafficking dysregulated DAT coding variant identified in an Attention Deficit Hyperactivity Disorder (ADHD) patient via constitutively activating Ack1. To address what mechanisms govern DAT’s post-endocytic fate, I took advantage of a small molecule labeling approach to directly couple fluorophore to the DAT surface population, and subsequently tracked DAT’s temporal-spatial post-endocytic itinerary in immortalized mesencephalic cells. Using this approach, I discovered that the retromer complex mediates DAT recycling and is required to maintain DAT surface levels via a DAT C-terminal PDZ-binding motif. Taken together, these findings shed considerable new light on DAT trafficking mechanisms, and pave the way for future studies examining the role of regulated DAT trafficking in neuropsychiatric disorders.
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Wu, Sijia. "Novel Mechanisms Regulating Dopamine Transporter Endocytic Trafficking: Ack1-Controlled Endocytosis And Retromer-Mediated Recycling." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/887.
Full textAbstract:
Dopamine transporters (DAT) facilitate high-affinity presynaptic dopamine (DA) reuptake in the central nervous system, and are required to constrain extracellular DA levels and maintain presynaptic DAergic tone. DAT is the primary target for addictive and therapeutic psychostimulants, which require DAT binding to elicit reward. DAT availability at presynaptic terminals ensures its proper function, and is dynamically regulated by endocytic trafficking. My thesis research focused on two fundamental questions: 1) what are the molecular mechanisms that control DAT endocytosis? and 2) what are the mechanism(s) that govern DAT’s post-endocytic fate? Using pharmacological and genetic approaches, I discovered that a non-receptor tyrosine kinase, activated by cdc42 kinase 1 (Ack1), stabilizes DAT plasma membrane expression by negatively regulating DAT endocytosis. I found that stimulated DAT endocytosis absolutely requires Ack1 inactivation. Moreover, I was able to restore normal DAT endocytosis to a trafficking dysregulated DAT coding variant identified in an Attention Deficit Hyperactivity Disorder (ADHD) patient via constitutively activating Ack1. To address what mechanisms govern DAT’s post-endocytic fate, I took advantage of a small molecule labeling approach to directly couple fluorophore to the DAT surface population, and subsequently tracked DAT’s temporal-spatial post-endocytic itinerary in immortalized mesencephalic cells. Using this approach, I discovered that the retromer complex mediates DAT recycling and is required to maintain DAT surface levels via a DAT C-terminal PDZ-binding motif. Taken together, these findings shed considerable new light on DAT trafficking mechanisms, and pave the way for future studies examining the role of regulated DAT trafficking in neuropsychiatric disorders.
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Gao, Xin D. "Combining CRISPR-Cas9 and Proximity Labeling to Illuminate Chromatin Composition, Organization, and Regulation." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1053.
Full textAbstract:
A bacterial and archaeal adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), has recently been engineered for genome editing. This RNA-guided platform has simplified genetic manipulation and holds promise for therapeutic applications. However, off-target editing has been one of the major concerns of the commonly used Streptococcus pyogenes Cas9 (SpyCas9). Despite extensive enzyme engineering to reduce off-target editing of SpyCas9, we have turned to nature and uncovered a Cas9 ortholog from Neisseria meningitidis (Nme) with high fidelity. In the first part of my thesis, we have systematically characterized Nme1Cas9 for engineering mammalian genomes and demonstrated its high specificity by genome-wide off-targeting detection methods in vitro and in cellulo, and thus provided a new platform for accurate genome editing.
Due to its flexibility, CRISPR is becoming a versatile tool not only for genome editing, but also for chromatin manipulation. These alternative applications are possible because of the programmable targeting capacity of catalytically dead Cas9 (dCas9). In the second part of my thesis, we have combined dCas9 with the engineered plant enzyme ascorbate peroxidase (APEX2) to develop a proteomic method called dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST). Relying on the spatially restricted, fast biotin labeling of proteins near defined genomic loci, C-BERST enables the high-throughput identification of known telomere- and centromere- associated proteomes and novel factors. Furthermore, we have extended C-BERST to map the c-fos promoter and gained new insights regarding the dynamic transcriptional regulation process. Taken together, C-BERST can advance our understanding of chromatin regulators and their roles in nuclear and chromosome biology.
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Fagan, Rita R. "Rit2-Dependent Dopamine Transporter Endocytosis: Intrinsic Mechanism and In Vivo Impact." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1086.
Full textAbstract:
Dopamine (DA) governs movement, sleep, reward, and cognition. The presynaptic dopamine transporter (DAT), clears released DA, controlling DA signaling and homeostasis. Genetic DAT ablation causes hyperactivity, sleep reduction, and altered psychostimulant response. DAT surface expression is dynamic; DAT constitutively internalizes and recycles to and from the plasma membrane, and acute PKC activation stimulates DAT endocytosis. Cell line experiments demonstrated that PKC-stimulated DAT endocytosis requires Ack1 inactivation and the GTPase, Rit2. How Rit2 controls PKC-dependent DAT internalization, or whether regulated DAT endocytosis impacts behavior, is unknown. Here, I present data supporting that PKC activation stimulates Rit2/DAT dissociation, mediated by the DAT N-terminus. Further, Ack1 and Rit2 function independently to facilitate PKC-stimulated DAT internalization. Moreover, PKC-stimulated DAT endocytosis was limited to ventral striatum in ex vivo slice preparations, and required Rit2. Our lab previously demonstrated that certain DA-dependent behaviors required DAergic Rit2 in mice, however whether this was due to perturbed PKC-stimulated DAT internalization, or DAT-independent Rit2 function(s) remains untested. To address this, I turned to Drosophila and its Rit2 homolog Ric. I found that Ric and dDAT proteins interact in cell lines, and that constitutively active Ric (RicQ117L) increased dDAT function in cultured cells and ex vivo whole fly brains. However, neither DAergic Ric knockdown nor RicQ117L altered overall locomotion or sleep, suggesting that these fundamental behaviors do not require DAergic Ric. Together, these results expand our understanding of intrinsic mechanisms controlling DAT endocytosis, and their impact on behavior.
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Carr, Michael I. "The Role of MDM2 Phosphorylation in P53 Responses to DNA Damage and Tumor Suppression: A Dissertation." eScholarship@UMMS, 2016. http://escholarship.umassmed.edu/gsbs_diss/847.
Full textAbstract:
The p53 tumor suppressor protein is upregulated in response to DNA damage and other stress signals. The upregulation of p53 involves freeing it from negative regulation imposed by Mdm2 and MdmX (Mdm4). Accumulating evidence indicates that phosphorylation of Mdm proteins by different stress-activated kinases such as ATM or c-Abl significantly impacts p53 functions. We have previously shown that ATM phosphorylation of Mdm2 Ser394 is required for robust p53 stabilization and activation following DNA damage.
This dissertation describes in vivo examination of the mechanism by which Mdm2 Ser394 phosphorylation impacts p53 activities and its contribution to suppression of oncogene and DNA damage-induced tumors. We determine that phosphorylation of Mdm2 Ser394 regulates p53 activity by modulating Mdm2 stability and paradoxically delays Myc-driven lymphomagenesis while increasing lymphomagenesis in sub-lethally irradiated mice. c-Abl phosphorylates the residue neighboring Mdm2 Ser394, Mdm2 Tyr393.
This dissertation describes the generation of a novel Mdm2Y393F mutant mouse to determine if c-Abl phosphorylation of Mdm2 regulates p53-mediated DNA damage responses or tumor suppression in vivo. Mdm2Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2Y393F/S394A mice and Mdm2S394A mice display similar phenotypes.
The studies presented herein further our understanding of the mechanisms by which DNA damage-associated kinases stabilize and activate p53, and influence p53-dependent responses and tumor suppression. A better understanding of the in vivo effects of Mdm2 phosphorylation may facilitate the development of novel therapeutics capable of stimulating p53 anti-tumor activity or alleviating p53- dependent toxicities in non-malignant tissues.
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Haines, Ricci. "The Role of Argininosuccinate Synthase Serine 328 Phosphorylation in Nitric Oxide Production." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4061.
Full textAbstract:
Until recently, the main mechanism of argininosuccinate synthase (AS) regulation was described to exist mainly at the level of transcription. Transcriptional regulation of AS has been shown to be coordinate with eNOS in response to shear stress, hypoxia, tumor necrosis factor á (TNF-á), and PPAR ã agonist troglitizone. However, it is now understood that one level of NO regulation is cellular control of arginine availability to eNOS via post-translational modifications of AS such as phosphorylation. The purpose of this investigation was to determine under what conditions AS is phosphorylated at S328, identify the pathway that AS phosphorylation at S328 plays a role, and how phosphorylation affects AS function in endothelial cells. We developed a phospho-specific antibody directed against pS328 AS and assayed for increases or decreases in phosphorylation relative to physiological factors. We found that AS phosphorylation at S328 occurred when endothelial cells were stimulated with physiological factors that stimulate nitric oxide production through calcium-dependent stimulation of eNOS. Furthermore, by utilizing kinase inhibitors and kinase knockdown experiments, we showed that phosphorylation at S328 significantly decreased when PKCá was knocked down, suggesting that S328 phosphorylation of AS is involved in PKCá signaling. In addition, by confocal microscopy, immunoprecipitation, and membrane fractionation, we showed that phosphorylation at S328 of AS promotes its co-localization with eNOS in the perinuclear region. These findings describe a novel pathway involving AS regulation of nitric oxide production, and may serve as a novel drug target in the restoration of vascular nitric oxide homeostasis.
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Gruber, Claudia. "Investigation into the regulatory mechanism of BRCA2 stability." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:e69ab649-f955-48d2-a7c5-48b65f15df45.
Full textAbstract:
Inherited mutations in the BRCA2 gene predispose individuals to the development of breast and ovarian cancers. The BRCA2 protein plays a fundamental role in the repair of DNA double strand breaks by homologous recombination (HR). BRCA2 mediates the recruitment of the RAD51 recombinase to DNA damage sites, which in turn promotes homologous pairing and strand exchange during HR. It has been reported that increased BRCA2 mRNA levels correlate with poor cancer prognosis, and recently it has been shown that increased levels of BRCA2 suppress HR. As HR is regulated through the cell cycle and can only be employed during S and G2 phases of the cell cycle, in this study, the cell cycle-dependent regulation of BRCA2, as a key player of HR, was investigated. In this study I report that BRCA2 stability is regulated by the ubiquitin-proteasome system (UPS), which has become increasingly evident as an important regulator of DNA repair. In line with this, I found that BRCA2 can be ubiquitylated in vivo and that it interacts with proteins of the UPS. Interestingly, I observed that BRCA2 levels and its ubiquitylation status change during the cell cycle. Using a siRNA-based approach, I identified a candidate E3 ubiquitin ligase, the SCFFBXW7 complex, which is also a known major cell cycle regulator. siRNA-mediated knockdown of FBXW7 led to stabilization of BRCA2 and overexpression of FBXW7 resulted in BRCA2 ubiquitylation in vivo. Furthermore, I have refined the regions that the SCFFBXW7 interacts with on BRCA2, which likely occurs in a phosphorylation-dependent manner. Taken together, these observations suggest that BRCA2 stability is regulated by the UPS in a cell cycle-dependent manner, which may be an important regulatory mechanism for BRCA2 function.
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Peterneva, Ksenia. "Determining the mechanism of pathogenesis of mucolipidosis type IV and related lysosomal storage disorders for development of novel therapies." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:321b1da6-0033-4230-b047-b643e5ea3e60.
Full textAbstract:
Mucolipidosis type IV (MLIV) is a rare, autosomal recessive, neurodegenerative, lysosomal storage disorder. MLIV is caused by mutations in a gene (MCOLN1) encoding a TRP channel family member known as Mucolipin 1 or TRPML1. TRPML1 is a lysosomal transmembrane protein that appears to be required for normal lysosomal pH regulation, recycling of molecules and membrane reorganisation including lysosomal biogenesis, fusion and exocytosis. The exact function of the channel is unknown but it is permeable to multiple ions including Ca2+, Na+ and K+, possibly also Fe2+ and Zn2+. How normal TRPML1 function regulates lysosomal processes is not clearly understood. Mutations in the MCOLN1 gene can lead to complete loss of TRPML1 function, partial loss of function or mislocalisation, all of which lead to lysosomal dysfunction, lysosomal lipid storage and ultimately neurodegeneration. The disease processes that lead to neurodegeneration are poorly understood and at present no therapy exists for MLIV. We have discovered that TRPML1 results in regulating lysosomal Ca2+ homeostasis that is the opposite of the Ca2+ dysregulation associated with Niemann-Pick type C disease (NPC). Our findings indicate that disrupted function of TRPML1 leads to enhanced Ca2+ release via the NAADP receptor, recently shown to be the lysosomal two-pore channel TPC2. This indicates that TRPML1 is not the NAADP receptor as suggested by others, indeed NAADP mediated Ca2+ release is enhanced with multiple NAADP induced lysosomal Ca2+ release events occurring in TRPML1 null cells compared to single releases in normal cells. This phenotype appears to be responsible for the cellular dysfunction associated with MLIV disease cells, enhanced lysosomal fusion, defective endocytosis and potentially even altered lysosomal pH. Several of these phenotypes are normalised by the NAADP receptor specific antagonist Ned-19. These findings illustrate that the NAADP receptor is central to MLIV disease pathology and may be a novel candidate for disease therapy.
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Hu, Yu-Jie. "Roles of Protein Arginine Methyltransferase 7 and Jumonji Domain-Containing Protein 6 in Adipocyte Differentiation: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/797.
Full textAbstract:
Regulation of gene expression comprises a wide range of mechanisms that control the abundance of gene products in response to environmental and developmental changes. These biological processes can be modulated by posttranslational modifications including arginine methylation. Among the enzymes that catalyze the methylation, protein arginine methyltransferase 7 (PRMT7) is known to modify histones to repress gene expression. Jumonji domain-containing protein 6 (JMJD6) is a putative arginine demethylase that potentially antagonize PRMT7. However, the biological significance of these enzymes is not well understood. This thesis summarizes the investigation of both PRMT7 and JMJD6 in cell culture models for adipocyte differentiation. The results suggest that PRMT7 is not required for the differentiation, whereas JMJD6 is necessary for the differentiation by promoting the expression of the lineage determining transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancerbinding proteins (C/EBPs). The underlying mechanisms by which JMJD6 regulate differentiation involve transcriptional and post-transcriptional control of gene expression. Unexpectedly, the adipogenic function of JMJD6 is independent of its enzymatic activity. Collectively, the present research reveals a novel role of JMJD6 in gene regulation during the differentiation of adipocytes.
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Hu, Yu-Jie. "Roles of Protein Arginine Methyltransferase 7 and Jumonji Domain-Containing Protein 6 in Adipocyte Differentiation: A Dissertation." eScholarship@UMMS, 2010. http://escholarship.umassmed.edu/gsbs_diss/797.
Full textAbstract:
Regulation of gene expression comprises a wide range of mechanisms that control the abundance of gene products in response to environmental and developmental changes. These biological processes can be modulated by posttranslational modifications including arginine methylation. Among the enzymes that catalyze the methylation, protein arginine methyltransferase 7 (PRMT7) is known to modify histones to repress gene expression. Jumonji domain-containing protein 6 (JMJD6) is a putative arginine demethylase that potentially antagonize PRMT7. However, the biological significance of these enzymes is not well understood. This thesis summarizes the investigation of both PRMT7 and JMJD6 in cell culture models for adipocyte differentiation. The results suggest that PRMT7 is not required for the differentiation, whereas JMJD6 is necessary for the differentiation by promoting the expression of the lineage determining transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancerbinding proteins (C/EBPs). The underlying mechanisms by which JMJD6 regulate differentiation involve transcriptional and post-transcriptional control of gene expression. Unexpectedly, the adipogenic function of JMJD6 is independent of its enzymatic activity. Collectively, the present research reveals a novel role of JMJD6 in gene regulation during the differentiation of adipocytes.
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Fogeron, Marie-Laure. "Development of a wheat germ cell-free expression system for the production, the purification and the structural and functional characterization of eukaryotic membrane proteins : application to the preparation of hepatitis C viral proteins." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10081/document.
Full textAbstract:
Alors que 30% du génome code pour des protéines membranaires, moins de 3% des structures protéiques dans la Protein Data Bank correspondent à ces protéines. En raison de leur nature hydrophobe, les protéines membranaires sont en effet très difficiles à produire dans des systèmes d'expression classique en cellules, notamment en bactéries. L'étude structurale des protéines membranaires du virus de l'hépatite C (VHC) sous forme entière et native a donc été pendant longtemps entravée. Le VHC est un virus à ARN positif dont le complexe de réplication est basé sur un réarrangement spécifique des membranes induit par l'action concertée de plusieurs protéines non structurales du virus dont NS2, NS4B et NS5A. La structure tridimensionnelle et le rôle de ces protéines dans la réplication virale sont encore mal connus. Pour surmonter les limitations qui empêchent leurs études structurales et fonctionnelles, un système d'expression acellulaire à base d'extrait de germe de blé a été développé avec succès, permettant la production des protéines NS2, NS4B et NS5A entières directement sous une forme solubilisée en présence de détergent. Ces protéines membranaires sont produites et purifiées par chromatographie d'affinité dans des quantités de l'ordre du milligramme. Des analyses par filtration sur gel indiquent que les échantillons obtenus sont homogènes. De plus, des analyses structurales par dichroïsme circulaire montrent que les protéines produites dans ce système sont bien repliées. Leur reconstitution dans des lipides est en cours d'optimisation. Le but ultime est en effet de déterminer leur structure par RMN du solide dans un environnement lipidique mimant l'environnement natifWhile 30% of the genome encodes for membrane proteins, less than 3% of protein structures in the Protein Data Bank correspond to such proteins. Due to their hydrophobic nature, membrane proteins are indeed notoriously difficult to express in classical cell-based protein expression systems. The structural study of the membrane proteins of hepatitis C virus (HCV) in their full-length and native form has therefore been for long time hampered. HCV is a positive-strand RNA virus building its replication complex on a specific membrane rearrangement (membranous web), which serves as a scaffold for the HCV replicase, and is induced by the concerted action of several HCV non-structural proteins including NS2, NS4B and NSSA. The knowledge of the three- dimensional structure of these proteins and their role in virus replication is still limited. To overcome the limitations that prevent the structural and functional studies of these proteins, a wheat germ cell-free protein expression system has been developed. A production protocol was designed which allows us to directly obtain membrane proteins in a soluble form by adding detergent during the in vitro protein synthesis. A large number of mainly viral proteins were successfully expressed, and full protocols were developed for the full-length NS2, NS4B and NSSA proteins. These membrane proteins were produced and purified by affinity chromatography using a Strep-tag II in the milligram range. These protein samples are homogenous, as shown by gel filtration analysis. Moreover, structural analyses by circular dichroism showed that the proteins produced in the wheat germ cell-free system are well folded. Reconstitution of these proteins in lipids is currently under optimization. The ultimate goal is to determine their structure by solid-state NMR in a native-like membrane lipids environment
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Chen, Hsiuyi V. "Systematic Dissection of Roles for Chromatin Regulators in Dynamics of Transcriptional Response to Stress in Yeast: A Dissertation." eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/808.
Full textAbstract:
The following work demonstrates that chromatin regulators play far more pronounced roles in dynamic gene expression than they do in steady-state. Histone modifications have been associated with transcription activity. However, previous analyses of gene expression in mutants affecting histone modifications show limited alteration. I systematically dissected the effects of 83 histone mutants and 119 gene deletion mutants on gene induction/repression in response to diamide stress in yeast. Importantly, I observed far more changes in gene induction/repression than changes in steady-state gene expression. The extensive dynamic gene expression profile of histone mutants and gene deletion mutants also allowed me to identify specific interactions between histone modifications and chromatin modifiers. Furthermore, by combining these functional results with genome-wide mapping of several histone modifications in the same time course, I was able to investigate the correspondence between histone modification occurrence and function. One such observation was the role of Set1-dependent H3K4 methylation in the repression of ribosomal protein genes (RPGs) during multiple stresses. I found that proper repression of RPGs in stress required the presence, but not the specific sequence, of an intron, an element which is almost unique to this gene class in Saccharomyces cerevisiae. This repression may be related to Set1’s role in antisense RNA-mediated gene silencing. Finally, I found a potential role for Set1 in producing or maintaining uncapped mRNAs in cells through a mechanism that does not involved nuclear exoribonucleases. Thus, deletion of Set1 in xrn1Δ suppresses the accumulation of uncapped transcripts observed in xrn1Δ. These findings reveal that Set1, along with other chromatin regulators, plays important roles in dynamic gene expression through diverse mechanisms and thus provides a coherent means of responding to environmental cues.
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Huang, Nai-Jia. "The role of TRIM39 in cell cycle and apoptosis." Diss., 2013. http://hdl.handle.net/10161/8245.
Full textAbstract:
Within individual cells, the opposing processes of proliferation and apoptosis are precisely regulated. When this regulatory balance is interrupted, cells may become abnormal or even transformed. Understanding how to reverse or avoid these detrimental transformative processes begins with an intimate knowledge of the processes governing the cell cycle and apoptosis. Cell proliferation is governed by the cell cycle machinery. The cell cycle is driven by Cyclin-dependent kinase (Cdk) activity, which is dependent on the availability of specific Cyclin binding partners. The amount of available Cyclin is tightly controlled by a ubiquitin ligase protein complex called the anaphase promoting complex/cyclosome (APC/C.) This complex mediates the timely ubiquitylation and degradation of cell cycle regulators in order to control mitotic exit, the G1/S transition and to respond to signals emanating from spindle assembly checkpoint.
Given the importance of the APC/C, cells develop many ways to regulate APC/C activity. Post-translational modifications of the APC/C have been shown to alter its functionality, and many pseudosubstrate-based inhibitors have been discovered. Moreover, inhibitors such as Emi1 and Emi2, have been showed to inhibit the APC/C through their own intrinsic ubiquitin E3 ligase activities. Utilizing the
To extend our observations regarding the role for TRIM39 in APC/C regulation, we investigated effects on the cell cycle via real-time imaging microscopy. We found cells arrest at G1/S in TRIM39 depleted RPE cells, a cell line which is commonly used for cell cycle analysis. This arrest phenotype is not observed in 293T, PC3 and H1299 cells which bear mutant p53 alleles. Further analysis showed that TRIM39 depleted RPE cells upregulate many genes that function downstream of p53 activity, such as the cdk inhibitor p21--thus, arresting cells at G1/S and reducing proliferation. The reduced growth can be rescued by p53 knockdown. Mechanistically, TRIM39 interacts with p53 and promotes destruction of p53 by ubiquitylation. This ubiquitylation is independent of the activity of the most intensively studied p53-directed E3 ligase, MDM2; depletion of both MDM2 and TRIM39 has a synergistic effect on p53 accumulation. This elevated p53 leads to more apoptosis in cancer cells bearing wildtype p53. Consequently, TRIM39 depletion might be employed as a combination treatment with MDM2 inhibitor, such as nutlin-3a, to stimulate tumor cell death.
In the thesis, we have found TRIM39 inhibits both the APC/C and p53. Both are essential regulators of cell cycle and apoptosis. Moreover, we have determined that the inhibitory activity of TRIM39 requires its E3 ligase activity. Future experiments will be directed towards investigating how TRIM39 protein stability and ligase activity are regulated to understand more fully the physiological situations in which TRIM39 is able to exert its ability to modulate the cell cycle and apoptosis. I will also discuss some preliminary data regarding changes in TRIM39 ligase activity induced by Chk1 and changes in TRIM39 protein abundance regulated by polo-like kinase 1(Plk1). Chk1 and Plk1 are essential kinases for cell cycle checkpoint and progression. Connecting Chk1 and Plk1 to TRIM39 may provide a more thorough understanding of TRIM39's ability to control the APC/C inhibition and p53 ubiquitylation in response to cell cycle or cell damage cues. Since the APC/C and p53 both can regulate cell cycle and apoptosis, further investigations into the involvement of TRIM39 in the life-or-death decision will be of great interest.
Dissertation
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O'Connell, Brett. "A study of rat skeletal muscle Troponin C isoforms." Thesis, 2005. https://vuir.vu.edu.au/15657/.
Full textAbstract:
The investigations described in this thesis were prompted by an overall interest in the phenomenon of Troponin C (TnC) polymorphism in mammalian skeletal muscle. Gaining
insights into this area of inquiry has been limited, in large part due to methodological problems associated with the identification of rat (a commonly used animal model for
studying mammalian skeletal muscle) TnC isoforms on SDS gels. Therefore, a method was devised for unambiguous identification of TnC isoforms in rat single muscle fibres.
This method, validated using rat skeletal muscle TnC isoforms purified for the first time as part of this study, was used in conjunction with myosin heavy chain (MHC) isoform based fibre-typing and Sr2+ -activation measurements to explore the relationship between MHC and TnC isoform composition in mammalian skeletal muscle at the single fibre level, and to revisit the controversial issue of the relationship between TnC isoform composition and fibre-type differences with respect to Sr2+ -activation characteristics.
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Barker, Megan. "Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae." Thesis, 2010. http://hdl.handle.net/1807/32660.
Full textAbstract:
N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications.
The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work.
To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs.