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Search Results: 1 - 10 of 304311 matches for " Steven J. Coultrap "
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Improving a Natural CaMKII Inhibitor by Random and Rational Design
Steven J. Coultrap,K. Ulrich Bayer
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0025245
Abstract: CaM-KIIN has evolved to inhibit stimulated and autonomous activity of the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) efficiently, selectively, and potently (IC50 ~100 nM). The CN class of peptides, derived from the inhibitory region of CaM-KIIN, provides powerful new tools to study CaMKII functions. The goal of this study was to identify the residues required for CaMKII inhibition, and to assess if artificial mutations could further improve the potency achieved during evolution.
A Significant but Rather Mild Contribution of T286 Autophosphorylation to Ca2+/CaM-Stimulated CaMKII Activity
Steven J. Coultrap, Kelsey Barcomb, K. Ulrich Bayer
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0037176
Abstract: Background Autophosphorylation of the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) at T286 generates partially Ca2+/CaM-independent “autonomous” activity, which is thought to be required for long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. A requirement for T286 autophosphorylation also for efficient Ca2+/CaM-stimulated CaMKII activity has been described, but remains controversial. Methodology/Principal Findings In order to determine the contribution of T286 autophosphorylation to Ca2+/CaM-stimulated CaMKII activity, the activity of CaMKII wild type and its phosphorylation-incompetent T286A mutant was compared. As the absolute activity can vary between individual kinase preparations, the activity was measured in six different extracts for each kinase (expressed in HEK-293 cells). Consistent with measurements on purified kinase (from a baculovirus/Sf9 cell expression system), CaMKII T286A showed a mildly but significantly reduced rate of Ca2+/CaM-stimulated phosphorylation for two different peptide substrates (to ~75–84% of wild type). Additional slower CaMKII autophosphorylation at T305/306 inhibits stimulation by Ca2+/CaM, but occurs only minimally for CaMKII wild type during CaM-stimulated activity assays. Thus, we tested if the T286A mutant may show more extensive inhibitory autophosphorylation, which could explain its reduced stimulated activity. By contrast, inhibitory autophosphorylation was instead found to be even further reduced for the T286A mutant under our assay conditions. On a side note, the phospho-T305 antibody showed some basal background immuno-reactivity also with non-phosphorylated CaMKII, as indicated by T305/306A mutants. Conclusions/Significance These results indicate that Ca2+/CaM-stimulated CaMKII activity is mildly (~1.2–1.3fold) further increased by additional T286 autophosphorylation, but that this autophosphorylation is not required for the major part of the stimulated activity. This indicates that the phenotype of CaMKII T286A mutant mice is indeed due to the lack of autonomous activity, as the T286A mutant showed no dramatic reduction in stimulated activity.
CaMKII Binding to GluN2B Is Differentially Affected by Macromolecular Crowding Reagents
Dayton J. Goodell, Tatiana A. Eliseeva, Steven J. Coultrap, K. Ulrich Bayer
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0096522
Abstract: Binding of the Ca2+/calmodulin(CaM)-dependent protein kinase II (CaMKII) to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B controls long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. Regulation of this interaction is well-studied biochemically, but not under conditions that mimic the macromolecular crowding found within cells. Notably, previous molecular crowding experiments with lysozyme indicated an effect on the CaMKII holoenzyme conformation. Here, we found that the effect of molecular crowding on Ca2+/CaM-induced CaMKII binding to immobilized GluN2B in vitro depended on the specific crowding reagent. While binding was reduced by lysozyme, it was enhanced by BSA. The ATP content in the BSA preparation caused CaMKII autophosphorylation at T286 during the binding reaction; however, enhanced binding was also observed when autophosphorylation was blocked. Importantly, the positive regulation by nucleotide and BSA (as well as other macromolecular crowding reagents) did not alleviate the requirement for CaMKII stimulation to induce GluN2B binding. The differential effect of lysozyme (14 kDa) and BSA (66 kDa) was not due to size difference, as both dextran-10 and dextran-70 enhanced binding. By contrast, crowding with immunoglobulin G (IgG) reduced binding. Notably, lysozyme and IgG but not BSA directly bound to Ca2+/CaM in an overlay assay, suggesting a competition of lysozyme and IgG with the Ca2+/CaM-stimulus that induces CaMKII/GluN2B binding. However, lysozyme negatively regulated binding even when it was instead induced by CaMKII T286 phosphorylation. Alternative modes of competition would be with CaMKII or GluN2B, and the negative effects of lysozyme and IgG indeed also correlated with specific or non-specific binding to the immobilized GluN2B. Thus, the effect of any specific crowding reagent can differ, depending on its additional direct effects on CaMKII/GluN2B binding. However, the results of this study also indicate that, in principle, macromolecular crowding enhances CaMKII binding to GluN2B.
Radiation Induces Acute Alterations in Neuronal Function
Peter H. Wu, Steven Coultrap, Chelsea Pinnix, Kurtis D. Davies, Ramesh Tailor, Kian K. Ang, Michael D. Browning, David R. Grosshans
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0037677
Abstract: Every year, nearly 200,000 patients undergo radiation for brain tumors. For both patients and caregivers the most distressing adverse effect is impaired cognition. Efforts to protect against this debilitating effect have suffered from inadequate understanding of the cellular mechanisms of radiation damage. In the past it was accepted that radiation-induced normal tissue injury resulted from a progressive reduction in the survival of clonogenic cells. Moreover, because radiation-induced brain dysfunction is believed to evolve over months to years, most studies have focused on late changes in brain parenchyma. However, clinically, acute changes in cognition are also observed. Because neurons are fully differentiated post-mitotic cells, little information exists on the acute effects of radiation on synaptic function. The purpose of our study was to assess the potential acute effects of radiation on neuronal function utilizing ex vivo hippocampal brain slices. The cellular localization and functional status of excitatory and inhibitory neurotransmitter receptors was identified by immunoblotting. Electrophysiological recordings were obtained both for populations of neuronal cells and individual neurons. In the dentate gyrus region of isolated ex vivo slices, radiation led to early decreases in tyrosine phosphorylation and removal of excitatory N-methyl-D-aspartate receptors (NMDARs) from the cell surface while simultaneously increasing the surface expression of inhibitory gamma-aminobutyric acid receptors (GABAARs). These alterations in cellular localization corresponded with altered synaptic responses and inhibition of long-term potentiation. The non-competitive NMDAR antagonist memantine blocked these radiation-induced alterations in cellular distribution. These findings demonstrate acute effects of radiation on neuronal cells within isolated brain slices and open new avenues for study.
Rank Functions of Fuzzy Greedoids  [PDF]
Steven J. Tedford
Open Journal of Discrete Mathematics (OJDM) , 2015, DOI: 10.4236/ojdm.2015.54006
Abstract: Fuzzy greedoids were recently introduced as a fuzzy set generalization of (crisp) greedoids. We characterize fuzzy languages which define fuzzy greedoids, give necessary properties and sufficient properties of the fuzzy rank function of a fuzzy greedoid, give a characterization of the rank function for a weighted greedoid, and discuss the rank closure of a fuzzy greedoid.
Oncoselectivity in Oncolytic Viruses against Colorectal Cancer  [PDF]
Steven J. Conrad, Karim Essani
Journal of Cancer Therapy (JCT) , 2014, DOI: 10.4236/jct.2014.513118

In humans colorectal cancer (CRC) is a significant cause of morbidity and mortality. New treatment options are urgently needed to supplement existing therapies. Replication-competent oncolytic viruses (RCOVs) for the treatment of cancerous tumorsin vivois a relatively new therapeutic modality with great but largely unrealized potential against CRC. In the context of oncolytic virus safety, oncoselectivity is an important criterion. It is at the conceptual intersection of viral replication strategy and tumor cell biology that RCOVs acquire their oncoselectivity, and thus their safety. Every aspect of tumor molecular biology which distinguishes it from normal, non-neoplastic cells is a potential target for exploitation. In the first section of this review we will provide an explanation of some of the successful and widely used strategies for improving oncoselectivity in wild-type viruses to make them more suitable as RCOVs. In the second section we will describe some of the characteristics of CRC biology which can be exploited to provide oncoselectivity against CRC. Throughout the review examples of successfully-engineered RCOVs which embody the approach or strategy under discussion are noted. By showing what has been done, we hope to highlight what is possible and what remains to be done to generate oncoselective RCOVs for use against CRC in humans.

Challenges in Managing Hospitalized HIV Infected Persons with Low Absolute CD4 and Preserved CD4 Percentage  [PDF]
Steven M. Bobula, Carl J. Fichtenbaum
World Journal of AIDS (WJA) , 2012, DOI: 10.4236/wja.2012.23031
Abstract: Background: HIV infected persons are at risk for opportunistic illnesses based upon severity of immune deficiency. Management is generally based upon the most recent absolute CD4 count. We hypothesized there is a group of patients with a low absolute CD4 count and preserved CD4 percentage that are at low risk of AIDS-related opportunistic illnesses (OI). Methods: A retrospective review of medical records in HIV-infected persons hospitalized from 2004-2006. Individuals without CD4 counts available within 180 days of admission and during hospitalization were excluded. Patients with a decrease in the absolute CD4 count during hospitalization and stable CD4 percentage were compared to the rest of the cohort. Appropriate management was defined using DHHS guidelines for the prevention and treatment of opportunistic illnesses in HIV infection. Results: 464 patients had 978 hospitalizations. In 221 hospitalizations (N = 161 patients) inpatient and outpatient CD4 counts were available. In 35 hospitalizations (N = 25 patients) the absolute CD4 count declined with stable CD4 percent (cases). Cases had an average decline in CD4 of –197 cells/mm3 compared to –5 cells/mm3 in the comparator group. 30% of comparators had AIDS defining OI's compared to none in the case group (p = 0.01). Management outside of DHHS guidelines was more common in cases compared to the comparator group (49% vs 30%, p = 0.048). The median length of stay was prolonged in cases with management outside guidelines compared to appropriately managed persons in the comparator group (7 days vs 3.5 days, p = 0.03). Conclusion: In persons on potent antiretroviral therapy, abrupt declines in absolute CD4 counts without an accompanying change in CD4 percentage are associated with a low risk of AIDS related opportunistic infection, a higher rate of in-patient management outside DHHS guidelines, and a more prolonged length of stay.
Concurrent Phenologies of Three Semiaquatic Bugs (Heteroptera: Gerridae, Veliidae) on a Small River in Central Illinois, USA
Steven J. Taylor
Psyche , 2009, DOI: 10.1155/2009/562471
Abstract: The phenology of three species of Gerroidea (Heteroptera), Metrobates hesperius Uhler (Gerridae), Rhagovelia oriander Parshley (Veliidae), and Rhematobates tenuipes Meinert (Gerridae), was studied on a river in central Illinois (USA). Metrobates hesperius was the most abundant species, and was active from mid-May through mid-October. It was bivoltine and overwintered as eggs. Matinig and oviposition of M. hesperius were observed in mid-July. Rhagovelia oriander was present from mid-May to mid-November. This species was bivoltine (or possibly trivoltine), overwintering as eggs. Rheumatobates tenuipes was not active until early August, and was present to mid-November and was univoltine. It overwinters as adults and possibly as nymphs, and may undergo an extended early season diapause. The three species occupied differing microhabitats and differed in periods of peak abundance, with M. hesperius being most abundant from mid-May through the first of August, and R. tenuipes being most abundant from early August to mid-November.
Limits of Calcium Clearance by Plasma Membrane Calcium ATPase in Olfactory Cilia
Steven J. Kleene
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005266
Abstract: Background In any fine sensory organelle, a small influx of Ca2+ can quickly elevate cytoplasmic Ca2+. Mechanisms must exist to clear the ciliary Ca2+ before it reaches toxic levels. One such organelle has been well studied: the vertebrate olfactory cilium. Recent studies have suggested that clearance from the olfactory cilium is mediated in part by plasma membrane Ca2+-ATPase (PMCA). Principal Findings In the present study, electrophysiological assays were devised to monitor cytoplasmic free Ca2+ in single frog olfactory cilia. Ca2+ was allowed to enter isolated cilia, either through the detached end or through membrane channels. Intraciliary Ca2+ was monitored via the activity of ciliary Ca2+-gated Cl? channels, which are sensitive to free Ca2+ from about 2 to 10 μM. No significant effect of MgATP on intraciliary free Ca2+ could be found. Carboxyeosin, which has been used to inhibit PMCA, was found to substantially increase a ciliary transduction current activated by cyclic AMP. This increase was ATP-independent. Conclusions Alternative explanations are suggested for two previous experiments taken to support a role for PMCA in ciliary Ca2+ clearance. It is concluded that PMCA in the cilium plays a very limited role in clearing the micromolar levels of intraciliary Ca2+ produced during the odor response.
The dynamic proteome of Lyme disease Borrelia
Steven J Norris
Genome Biology , 2006, DOI: 10.1186/gb-2006-7-3-209
Abstract: We have all experienced it: the 'deer in the headlights' sensation of dumbfounded wonderment and awe when confronted with our first genome sequence. This is a particularly likely response to the genome of the spirochete Borrelia burgdorferi and its relatives - spiral bacteria that are transmitted by deer ticks of the Ixodes ricinus group and that cause the chronic disease Lyme borreliosis in humans and other animals. Although there were previous indications of its unusual characteristics, no one anticipated that the 1.5 megabase genome of B. burgdorferi would contain an odd mixture of approximately 12 linear and 9 circular plasmids, as well as a 0.9 megabase linear chromosome [1,2]. The plasmids, which range from 5 to 56 kilobases, are present consistently in the strains examined so far and contain genes required for the spirochete's life cycle; these replicons thus could be considered 'mini-chromosomes', although the term plasmids is typically used for simplicity. The 833 predicted plasmid-encoded open reading frames (ORFs) include 454 hypothetical genes, many of which are members of 107 paralogous families of unknown function. The plasmids are also rife with pseudogenes, leading to the conclusion that the Lyme disease spirochete genome is 'in flux': that is, it is actively evolving [2]. The unique properties of the Borrelia proteome are now starting to be revealed with two recent studies of global protein expression. In the first approach, Jacobs et al. [3] compared the protein profiles of three strains of B. burgdorferi, while in the second, Nowalk et al. [4] examined the proteins present in the soluble and membrane-associated fractions of the bacterium.Lyme disease is a chronic disease marked by skin lesions, debilitating neurologic symptoms and arthritis. B. burgdorferi is the predominant cause of human Lyme borreliosis in North America, whereas B. burgdorferi and two related Borrelia species, B. garinii and B. afzelii, cause disease in Eurasia. These obligate
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