bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conformational Dynamics of the PH Domain in Phospholipases Cɛ and β may Contribute to Subfamily‐Specific Roles in Regulation
Authors: Garland‐Kuntz Elisabeth E , Vago Frank S. , Sieng Monita , Van Camp Michelle M. , Blaine Arryn T. , Corpstein Clairissa , Chakravarthy Srinivas , Jiang Wen , Lyon Angeline M. 
Published: June 2021

Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce second messengers, including inositol‐1,4,5‐triphosphate (IP3) and diacylgycerol (DAG), which increase intracellular calcium and activate protein kinase C (PKC), respectively. PLCɛ contributes to cardiac hypertrophy and contractility, as well as to oncogenic and inflammatory signaling pathways following activation of G protein‐coupled receptors and receptor tyrosine kinases. PLCɛ shares a conserved core with the PLC superfamily, but the roles of individual domains in regulation of activity and membrane binding have not been established. We used functional assays to show that the PLCɛ PH domain significantly increases basal lipase activity, but is dispensable for stability. We provide the first structural insights into domain organization of PLCɛ using small‐angle X‐ray scattering (SAXS) and electron microscopy (EM) to reveal that the PH domain is conformationally heterogeneous in solution. Comparisons of the PLCɛ solution structure to that of the closely‐related PLCβ enzyme demonstrate that the PLCβ PH domain is also mobile in solution, in contrast to previously reported crystal structures. We propose that the dynamic nature of the PLC PH domain and resulting conformational heterogeneity contributes to subfamily‐specific differences in activity and regulation by G proteins. We are now using cryo‐EM to expand on these findings and obtain higher resolution structures.Support or Funding InformationThis work is supported by the Purdue Center for Cancer Research, AHA grant 16SDG29930017, NIH NLHBI 1R01HL141076‐01 to A.M.L., and Purdue College of Science Staff and Administrative & Professional Staff Advisory Council Professional Development awards to E.E.G. SAXS experiments used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. This project was supported by grant 9 P41 GM103622 from the National Institute of General Medical Sciences of the National Institutes of Health. Use of the Pilatus 3 1M detector was provided by grant 1S10OD018090‐01 from NIGMS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unraveling signaling mechanisms regulating cluster‐based metastasis
Authors: Cheung Kevin , Wrenn Emma D , Moore Breanna , Yamamoto Ami , McBirney Margaux , Greenwood Erin , Thomas Aaron 
Published: June 2021

There is mounting evidence that tumor cells can migrate and metastasize as cell clusters. The presence of circulating tumor cell clusters is associated with early relapse and death in many cancer types. Experimentally, clustering of tumor cells greatly increases colonizing potential, but the molecular mechanisms explaining this phenomenon are largely unknown. We performed time‐lapse imaging and genome wide transcription studies in mouse and human breast tumor organoids. This revealed that clustering of tumor cells rapidly induces expression of multiple EGFR pathway genes and increased auto‐phosphorylation of EGFR. Interestingly, pEGFR colocalized with multiple EGFR ligands at cell‐cell contacts, suggesting that clusters' EGFR activation may be due to increased ligand binding. Consistent with this hypothesis, RNAi knockdowns of multiple individual ligands reduced tumor organoid growth by 40 to 95% of control. Downstream of receptor activation, expression of EGFR effector pERK was increased in clusters. The most significantly cluster‐upregulated transcription factor was the ERK‐activated oncogene Fra1. Fra1 knockdown reduced growth as did ERK inhibition. Using patient breast cancer samples, including primary and metastatic tumors, we found that Fra1 expression was highly upregulated in human tumor cell clusters, supporting the human disease relevance of this pathway. Taken together, our studies show that tumor cell clusters activate EGFR‐ERK‐Fra1 growth signaling at cell‐cell contacts. We propose that disrupting this pathway could reduce metastasis and extend survival in cancer patients.Support or Funding InformationI am grateful for funding support from the Burroughs Wellcome Fund, Department of Defense, Susan G. Komen, Breast Cancer Research Foundation, and the V Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of inhibitors on oxygen permeability of wild type and knockout mouse red blood cells
Authors: Zhao Pan , Occhipinti Rossana , Boron Walter F 
Published: June 2021

We now recognize that the paradigm that all gases cross red blood cell (RBC) membranes by dissolving in and diffusing through the lipid phase is not correct. Work on human RBCs genetically deficient in aquaporin‐1 (AQP1) or the Rh complex (mainly RhAG) showed that these two proteins account for ~90% of membrane CO2 permeability (PM,CO2), and that 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) reduces PM,CO2. We used stopped‐flow (SF) analysis of hemoglobin (Hb) absorbance spectra as we mixed oxygenated RBCs with an extracellular O2 scavenger, and determined the rate constant of Hb deoxygenation (kHbO2). Because kHbO2 depends on Hb kinetics as well as diffusion of O2 to the extracellular space, we used a mathematical model to estimate effects on membrane O2 permeability (PM,O2). We examined the effects of DIDS and chloromercuribenzenesulfonate (pCMBS), agents that decrease PM,CO2. We find that, in RBCs from wild type (WT) mice, pCMBS reduces kHbO2 by 64% (est. PM,O2 by ~84%) and DIDS, by 41% (est. PM,O2 by ~67%). Because pCMBS and DIDS likely act by targeting membrane proteins (rather than lipids), we hypothesized that O2, at least in part, crosses RBC membranes via the same membrane proteins that conduct CO2. Therefore, we examined RBCs from mice genetically deficient in AQP1, RhAG, or both, ± inhibitors. Deletion of AQP1 lowers kHbO2 by 11% (est. PM,O2 by ~27%); RhAG, by 16% (est. PM,O2 by ~35%); the double knockout (dKO), by 33% (est. PM,O2 by ~59%). The combination dKO + pCMBS lowers kHbO2 (vs. WT without inhibitors) by 77% (est. PM,O2 by ~91%), whereas dKO + DIDS lowers kHbO2 by 52% (est. PM,O2 by ~76%). The effects of pCMBS on estimated PM,O2 are very similar when we compare dKO vs. RhAG−/−, or when we compare WT vs. AQP1−/−. Thus, the deletion of AQP1 may have little effect on the sensitivity of PM,O2 to pCMBS—consistent with the hypothesis that AQP1 conducts O2 only via pathways other than the mercury‐sensitive monomeric pores (e.g., via the hydrophobic central pore). In summary, our data suggest that AQP1 and RhAG account for nearly 60% of RBC O2 permeability, and point towards the existence of other O2 channels that account for at least 30%.Support or Funding InformationSupported by ONR N00014‐15‐1‐2060, ONR N00014‐16‐1‐2535 to WFB and NIH K01‐DK107787 to ROThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ketamine Blunts Sympathetic Nerve Activity Responses to a Cold Pressor test
Authors: Moralez Gilbert , Huang Mu , Cramer Matthew N , Hendrix Joseph M , Hinojosa‐Laborde Carmen , Ryan Kathy L , Fu Qi , Crandall Craig G 
Published: June 2021

Pain evokes increases in blood pressure, heart rate, and central sympathetic outflow, and the perception of pain increases sympathetic nerve activity in an intensity‐dependent manner. That said, the mechanisms underlying the perception of pain and the effects on sympathetic nerve activity are not well established. The purpose of this experiment was to examine the effects of the analgesic ketamine, an NMDA receptor antagonist that reduces pain perception, on pain perception and muscle sympathetic nerve activity (MSNA) during a painful stimulus. In seven healthy participants (2 males; age 30 ± 6 yrs., mass of 83 ± 5 kg) MSNA and pain perception (Visual Analog Scale for Pain) were assessed during a 2‐min cold pressor test with and without intravenous infusion of 20 mg of ketamine, a dose consistent with guidelines from the US Army's Committee on Tactical Combat Casualty Care for pain management on the battlefield. Prior to the cold pressor test, ketamine alone increased heart rate (P=0.020), mean arterial blood pressure (P <0.001), and MSNA from 22 ± 6 burst/min to 32 ± 7 bust/min (P<0.01). During the subsequent cold pressor test, ketamine both decreased pain perception (Visual Analog Scale for Painscore ‐ control: 69 ± 22; ketamine: 22 ± 23, P<0.001) and blunted the peak increase in MSNA (control: Δ22 ± 4 burst/min; ketamine: Δ8 ± 7 burst/min). The increases in blood pressure, heart rate and sympathetic nerve activity elicited by the administration of ketamine suggest that ketamine is acting centrally within the cardiovascular control centers. Furthermore, these data support previous findings demonstrating that sympathetic outflow to a painful stimulus is graded with the pain perception.Support or Funding InformationFunding: Department of Defense – US Army, W81XWH1820012This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hair Cell Development and Regeneration in Zebrafish Lacking the Cannabinoid Receptor2
Authors: Deida Tiffany Tossas , Colon Luis , Rodríguez Roberto , Santana Alexis , Behra Martine 
Published: June 2021

Hair cells (HCs) are mechanosensory receptors found in sensory epithelia in the vertebrate inner ear that detect sound and head movements. HCs are fragile and prone to damage from various causes like disease, aging, and noise pollution. Unlike mammals, lower vertebrates including birds, amphibians and fish can regenerate HCs. To understand the underlying molecular mechanisms of HCs regeneration, we study the lateral line (LL) in a powerful genetic and regenerative animal model: the zebrafish. The LL is a skin‐deep fish sensory organ that is evolutionarily linked to the vertebrate sensory epithelia of the inner ear. It is composed of stereotypical distributed sensory patches called neuromasts (NMs) which contain HCs that are highly similar in function, cellular structure and gene expression to HCs of the inner ear. LL‐HCs are deflected and activate by surrounding water movements. Using CRISPR‐Cas9 genome editing technology, our laboratory has previously generated a null allele of the cnr2 gene (cnr2upr1) which is encoding the cannabinoid receptor 2 (CB2). We found that cnr2 mRNA is highly expressed in LL‐HCs. So, we hypothesized that CB2 has a role in the development and the regeneration of LL‐HCs. We counted LL‐HCs at different developmental stages in wild type and homozygote mutant larvae (cnr2upr1/upr1) to see if their number is affected in the absence of a functional CB2 protein. Next, to trigger vigorous HCs regeneration, we synchronously ablated LL‐HCs by copper treatment of 5‐day post‐fertilization (dpf) wild type and homozygote animals. We counted regenerating LL‐HCs for 3‐day post‐treatment. We will present our preliminary results that will ultimately allow us to conclude on the involvement of CB2 in development and in regeneration of the LL and on a potential link between endocannabinoid signaling and sensory epithelia development and regeneration.Support or Funding InformationCRESTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of NMR to probe changes in the flexibility of acid‐stress chaperone HdeB from its inactive to active state
Authors: Abasi Lannah , Crowhurst Karin 
Published: June 2021

HdeB is an intrinsically disordered periplasmic chaperone essential to the acid resistance of numerous pathogenic bacteria. It, along with its homolog, HdeA, binds to vulnerable periplasmic proteins at low pH and prevents them from irreversibly aggregating in the acidic stomach. The activity of these two proteins is responsible for the survivability of pathogenic bacteria and resulting proliferation of dysentery. At physiological pH, HdeB is an inactive folded homodimer and remains a structurally unchanged, fully folded dimer in its active state (at pH 4–5), suggesting that the mechanism of chaperone activation is a result of changes in its dynamics (internal motions). The long‐term goal of this study is to gain insight into HdeB's unique chaperoning mechanism by characterizing its pH dependent changes in structure and dynamics. In order to achieve atomic level specificity and detect subtle conformational changes, we are utilizing Nuclear Magnetic Resonance (NMR) spectroscopy to compare HdeB's flexibility and structure in its inactive and active states at pH 6.0 and 4.5, respectively. Isotopically labeled HdeB was recombinantly expressed and purified. Subsequently, protein backbone and side chain dynamics were examined at pH 6 and pH 4.5 utilizing R2 and R1 relaxation experiments, which identify fast (ps–ns) time scale motions within the structure; this indicates local motions, such as methyl rotations. Additionally, intermediate (μs‐ms) time scale motions, which reflect global conformational changes and flexibility, were identified using CPMG relaxation dispersion experiments. Notable changes in dynamics were observed with activation. At pH 6.0, most residues with μs‐ms motions are clustered at the dimer binding interface (BC loop and helix B). With the transition to pH 4.5, μs‐ms motions expand within the dimer binding interface (W27, T33, G37, T40, K53) and extend beyond it (L16δ, I55δ). Furthermore, at pH 4.5, Helix B decreases in solvent protection, suggesting decreased burial and a loosening of the dimer binding interface. Since this helix is extremely hydrophobic, loosening exposes residues that are well suited to interacting with the exposed hydrophobic regions of an unfolding client protein, likely enabling interactions crucial to substrate binding. Interestingly, the largest changes with activation were observed near multiple titratable residues, making us suspect a protonation change. Continuing research will focus on experimentally determining acid dissociation constants to confirm this.Support or Funding InformationWe gratefully acknowledge the support of the NIH for research funds (SC3‐GM116745) and BUILD PODER for research support to L.A., as well as the NSF for funding the purchase of our NMR spectrometer (CHE‐1040134).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Arabidopsis Histone Deacetylase 5 is Required for Pseudomonas‐Induced Deacetylation of Histone H3 at Lysine 9
Authors: van Dijk Karin , Kim Panya , Hubbard Troy , Nguyen Linda , Guo Ming , Alfano Jim 
Published: June 2021

The gram‐negative plant pathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) depends on a type III secretion system (T3SS) to colonize plant tissue, and cause disease. The T3SS facilitates injection of virulence proteins called effector proteins (T3Es) directly into host cells. The T3Es have been shown to target components of plant immunity and are essential for Pto to cause disease. We have been interested in determining the extent that Pto T3Es are involved in modulating host chromatin to favor pathogenesis. Through Immunoblot and Chromatin Immunoprecipitation analyses we found a rapid deacetylation of host histone H3 lysine 9 (H3K9) along innate immunity related genes in response to Pto. This deacetylation was dependent on both T3Es and the host histone deacetylase HDA5. Moreover, Arabidopsis plants lacking HDA5 had an enhanced resistance to Pto infections and produced more callose in response to Pathogen Associated Molecular Pattern exposure. Moreover, through analyses of specific T3E Pto mutants and transient and transgenic expression of specific T3Es, we found that only a small subset of the T3Es are needed for the HDA5‐triggered deacetylation. Collectively our data show a Pto T3E‐induced deacetylation by HDA5 along immunity related genes in Arabidopsis.Support or Funding InformationThis work was supported by NSF award #1557417, NSF award # 0940177 and NIGMS award # 8P20GM103427This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EFFECTS OF ISOVOLEMIC HEMODILUTION ON TISSUE OXYGEN CONSUMPTION USING A HEMOGLOBIN‐BASED OXYGEN CARRIER AND HUMAN SERUM ALBUMIN
Authors: Song Bjorn Kyungsuck , Pittman Roland N. 
Published: June 2021

This microcirculatory study compared the effects on oxygen transport of two hemodilution fluids: HBOC‐201 (Biopure Corp., Cambridge, MA) a Hemoglobin‐Based Oxygen Carrier (HBOC), and 5.9% Human Serum Albumin (HSA) an iso‐oncotic non‐oxygen carrying colloid solution. Measurements using intravital microscopy were made on the spinotrapezius muscle of Sprague‐Dawley rats. Interstitial PO2 was measured using phosphorescence quenching microscopy, and recorded before and after isovolemic hemodilutions (HD) at hematocrits of 40% (baseline), 30% (moderate HD) and 15% (severe HD). Oxygen consumption (VO2) of the spinotrapezius muscle was derived from PO2 recordings following the rapid inflation of a Saran bag placed around the objective. When the bag was inflated, blood flow in the muscle was arrested and PO2 rapidly fell over several seconds; the rate of decline of PO2 was proportional to VO2. For moderate HD with HBOC‐201 interstitial PO2 did not change from baseline conditions (Hct~40%), while HD with HSA showed a decrease. For severe HD (Hct~15%) both PO2 and VO2 were significantly lower for the HSA group than for the HBOC‐201 group. These findings indicate that HBOC‐201 maintains both a higher PO2 and VO2 during hemodiluted states compared with a non‐oxygen carrying colloid solution (HSA).(Supported by Naval Medical Research Center, DOD and NIH grant HL18292)
Slow rate of neuromuscular activation contributes to impaired movement acceleration and peak power in mobility‐limited older adults
Authors: Clark David J , Patten Carolynn , Reid Kieran F , Carabello Robert , Phillips Edward M , Fielding Roger A 
Published: June 2021

Peak muscle power is a robust predictor of functional motor performance in older adults. A critical constituent of peak power is effective neuromuscular activation. This study investigated the relationship between pre‐movement activation rate, acceleration (Acc), and peak power (Ppeak) for a leg press task in healthy and mobility‐limited older adults.METHODS 19 middle aged healthy (MH, 40–55y, 9 male), 19 older healthy (OH, 70–85y, 14 male), and 14 older mobility‐limited (OML, 70–85y, 7 male, SPPB ≤ 9) adults participated. EMG was acquired from the quadriceps during 5 max effort leg press trials at 40% of 1‐rep max. EMG was rectified, smoothed, and normalized to peak from a maximal voluntary contraction (MVC). EMG from each trial was aligned to movement onset, then averaged and fit using linear regression to yield activation rate (slope).RESULTS OML demonstrated markedly lower activation rate (.13 %MVC/ms, p<.02) than MH (.24) and OH (.26). Similarly, Acc (4.4 m/s2, p<.01) and Ppeak (536W, p<.01) were impaired in OML relative to MH (6.5, 1057) and OH (5.8, 980). Across groups, activation rate was strongly related to Acc (R2=.66, p<.0001) and Ppeak (R2=.55, p<.001).CONCLUSION Attenuated rate of neuromuscular activation is associated with reduced movement acceleration and power production in mobility‐limited older adults.Supported by USDA 58‐1950‐7‐707and NIA RO1‐AG‐18844
The dependence of three P. abyssi inteins on extein sequence for efficient protein splicing
Authors: Kerrigan Adam M. , Powers Taryn L. , Dorval Deirdre M. , Reitter Julie N. , Mills Kenneth V. 
Published: June 2021

Protein splicing is catalyzed by an intervening polypeptide, called an intein. Inteins facilitate their own excision from the flanking polypeptides, called exteins, as well as the ligation of the exteins. Three inteins interrupt the ribonucleotide reductase of the extreme thermophile Pyrococcus abyssi. We have separately over‐expressed each intein in E. coli as a fusion protein with flanking affinity domains. By site directed mutagenesis, we have altered the sequences flanking each intein to determine the flexibility of the intein insertion site. We have also made mutations to residues suspected of catalyzing the splicing steps in each intein, and plan to study the influence of these mutations on the rate and extent of splicing.This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.
Molecular dynamics simulation of the NikR protein: Equilibrium conformational fluctuations reveal inter‐domain allosteric communication pathways
Authors: Bradley Michael John , Chivers Peter , Baker Nathan 
Published: June 2021

The Ni2+ and DNA binding protein NikR is involved in nickel regulation in Escherichia coli through transcriptional repression of the NikABCDE nickel permease. NikR is a homotetramer and each chain contains both a DNA binding ribbon‐helix‐helix (RHH) domain and a Ni2+ binding regulatory ACT (aspartokinase, chorismate mutase, TyrA) fold. In this study we have utilized an equilibrium molecular dynamics (MD) simulation in order to explore the conformational dynamics of the NikR tetramer and determine important residue interactions within and between the RHH and ACT domains. Using two different correlation measures based on fluctuations in atomic position and non‐covalent bonding, we have defined a series of residue interactions that describe an allosteric communication pathway between the Ni2+ and DNA binding sites. Several of the residues identified by our analyses have been shown experimentally to be important for NikR function. An additional subset of the selected residues structurally connects experimentally important residues and may help coordinate allosteric communication between the ACT and RHH domains. Site‐directed mutagenesis of E. coli NikR and subsequent biophysical characterization will reveal the role of these residues in allosteric activation of this regulatory protein.Support: NSF grant MCB‐0520877, NSF Graduate Research Fellowship to MJB