Browsing by Subject "Hydrogen-ion concentration"

Now showing 1 - 6 of 6
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    CDMS Analysis of Intact 19S, 20S, 26S, and 30S Proteasomes: Evidence for Higher-Order 20S Assemblies at a Low pH†
    (American Chemical Society, 2023) Anthony, Adam J.; Gautam, Amit K. S.; Miller, Lohra M.; Ma, Yiran; Hardwick, Anya G.; Sharma, Anu; Ghatak, Subhadip; Matouschek, Andreas; Jarrold, Martin F.; Clemmer, David E.; Surgery, School of Medicine
    Charge detection mass spectrometry (CDMS) was examined as a means of studying proteasomes. To this end, the following masses of the 20S, 19S, 26S, and 30S proteasomes from Saccharomyces cerevisiae (budding yeast) were measured: m(20S) = 738.8 ± 2.9 kDa, m(19S) = 926.2 ± 4.8 kDa, m(26S) = 1,637.0 ± 7.6 kDa, and m(30S) = 2,534.2 ± 10.8 kDa. Under some conditions, larger (20S)x (where x = 1 to ∼13) assemblies are observed; the 19S regulatory particle also oligomerizes, but to a lesser extent, forming (19S)x complexes (where x = 1 to 4, favoring the x = 3 trimer). The (20S)x oligomers are favored in vitro, as the pH of the solution is lowered (from 7.0 to 5.4, in a 20 mM ammonium acetate solution) and may be related to in vivo proteasome storage granules that are observed under carbon starvation. From measurements of m(20S)x (x = 1 to ∼13) species, it appears that each multimer retains all 28 proteins of the 20S complex subunit. Several types of structures that might explain the formation of (20S)x assemblies are considered. We stress that each structural type [hypothetical planar, raft-like geometries (where individual proteasomes associate through side-by-side interactions); elongated, rodlike geometries (where subunits are bound end-to-end); and geometries that are roughly spherical (arising from aggregation through nonspecific subunit interactions)] is highly speculative but still interesting to consider, and a short discussion is provided. The utility of CDMS for characterizing proteasomes and related oligomers is discussed.
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    Characterization of intrinsically disordered proteins with electrospray ionization mass spectrometry: conformational heterogeneity of alpha-synuclein
    (Wiley, 2010-02-15) Frimpong, Agya K.; Abzalimov, Rinat R.; Uversky, Vladimir N.; Kaltashov, Igor A.; Medicine, School of Medicine
    Conformational heterogeneity of alpha-synuclein was studied with electrospray ionization mass spectrometry by analyzing protein ion charge state distributions, where the extent of multiple charging reflects compactness of the protein conformations in solution. Although alpha-synuclein lacks a single well-defined structure under physiological conditions, it was found to sample four distinct conformational states, ranging from a highly structured one to a random coil. The compact highly structured state of alpha-synuclein is present across the entire range of conditions tested (pH ranging from 2.5 to 10, alcohol content from 0% to 60%), but is particularly abundant in acidic solutions. The only other protein state populated in acidic solutions is a partially folded intermediate state lacking stable tertiary structure. Another, more compact intermediate state is induced by significant amounts of ethanol used as a co-solvent and appears to represent a partially folded conformation with high beta-sheet content. Protein dimerization is observed throughout the entire range of conditions tested, although only acidic solutions favor formation of highly structured dimers of alpha-synuclein. These dimers are likely to present the earliest stages in protein aggregation leading to globular oligomers and, subsequently, protofibrils
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    Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic
    (American Society for Microbiology, 2017-02-28) Mulye, Minal; Samanta, Dhritiman; Winfree, Seth; Heinzen, Robert A.; Gilk, Stacey D.; Department of Microbiology & Immunology, IU School of Medicine
    Coxiella burnetii is an intracellular bacterial pathogen and a significant cause of culture-negative endocarditis in the United States. Upon infection, the nascent Coxiella phagosome fuses with the host endocytic pathway to form a large lysosome-like vacuole called the parasitophorous vacuole (PV). The PV membrane is rich in sterols, and drugs perturbing host cell cholesterol homeostasis inhibit PV formation and bacterial growth. Using cholesterol supplementation of a cholesterol-free cell model system, we found smaller PVs and reduced Coxiella growth as cellular cholesterol concentration increased. Further, we observed in cells with cholesterol a significant number of nonfusogenic PVs that contained degraded bacteria, a phenotype not observed in cholesterol-free cells. Cholesterol had no effect on axenic Coxiella cultures, indicating that only intracellular bacteria are sensitive to cholesterol. Live-cell microscopy revealed that both plasma membrane-derived cholesterol and the exogenous cholesterol carrier protein low-density lipoprotein (LDL) traffic to the PV. To test the possibility that increasing PV cholesterol levels affects bacterial survival, infected cells were treated with U18666A, a drug that traps cholesterol in lysosomes and PVs. U18666A treatment led to PVs containing degraded bacteria and a significant loss in bacterial viability. The PV pH was significantly more acidic in cells with cholesterol or cells treated with U18666A, and the vacuolar ATPase inhibitor bafilomycin blocked cholesterol-induced PV acidification and bacterial death. Additionally, treatment of infected HeLa cells with several FDA-approved cholesterol-altering drugs led to a loss of bacterial viability, a phenotype also rescued by bafilomycin. Collectively, these data suggest that increasing PV cholesterol further acidifies the PV, leading to Coxiella death.IMPORTANCE The intracellular Gram-negative bacterium Coxiella burnetii is a significant cause of culture-negative infectious endocarditis, which can be fatal if untreated. The existing treatment strategy requires prolonged antibiotic treatment, with a 10-year mortality rate of 19% in treated patients. Therefore, new clinical therapies are needed and can be achieved by better understanding C. burnetii pathogenesis. Upon infection of host cells, C. burnetii grows within a specialized replication niche, the parasitophorous vacuole (PV). Recent data have linked cholesterol to intracellular C. burnetii growth and PV formation, leading us to further decipher the role of cholesterol during C. burnetii-host interaction. We observed that increasing PV cholesterol concentration leads to increased acidification of the PV and bacterial death. Further, treatment with FDA-approved drugs that alter host cholesterol homeostasis also killed C. burnetii through PV acidification. Our findings suggest that targeting host cholesterol metabolism might prove clinically efficacious in controlling C. burnetii infection.
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    Involvement of Intermediate Sulfur Species in Biological Reduction of Elemental Sulfur under Acidic, Hydrothermal Conditions
    (American Society for Microbiology, 2013) Boyd, Eric S.; Druschel, Gregory K.; Earth and Environmental Sciences, School of Science
    The thermoacidophile and obligate elemental sulfur (S(8)(0))-reducing anaerobe Acidilobus sulfurireducens 18D70 does not associate with bulk solid-phase sulfur during S(8)(0)-dependent batch culture growth. Cyclic voltammetry indicated the production of hydrogen sulfide (H(2)S) as well as polysulfides after 1 day of batch growth of the organism at pH 3.0 and 81°C. The production of polysulfide is likely due to the abiotic reaction between S(8)(0) and the biologically produced H(2)S, as evinced by a rapid cessation of polysulfide formation when the growth temperature was decreased, inhibiting the biological production of sulfide. After an additional 5 days of growth, nanoparticulate S(8)(0) was detected in the cultivation medium, a result of the hydrolysis of polysulfides in acidic medium. To examine whether soluble polysulfides and/or nanoparticulate S(8)(0) can serve as terminal electron acceptors (TEA) supporting the growth of A. sulfurireducens, total sulfide concentration and cell density were monitored in batch cultures with S(8)(0) provided as a solid phase in the medium or with S(8)(0) sequestered in dialysis tubing. The rates of sulfide production in 7-day-old cultures with S(8)(0) sequestered in dialysis tubing with pore sizes of 12 to 14 kDa and 6 to 8 kDa were 55% and 22%, respectively, of that of cultures with S(8)(0) provided as a solid phase in the medium. These results indicate that the TEA existed in a range of particle sizes that affected its ability to diffuse through dialysis tubing of different pore sizes. Dynamic light scattering revealed that S(8)(0) particles generated through polysulfide rapidly grew in size, a rate which was influenced by the pH of the medium and the presence of organic carbon. Thus, S(8)(0) particles formed through abiological hydrolysis of polysulfide under acidic conditions appeared to serve as a growth-promoting TEA for A. sulfurireducens.
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    Maternal Oxygen Supplementation Compared With Room Air for Intrauterine Resuscitation: A Systematic Review and Meta-analysis
    (American Medical Association, 2021) Raghuraman, Nandini; Temming, Lorene A.; Doering, Michelle M.; Stoll, Carolyn R.; Palanisamy, Arvind; Stout, Molly J.; Colditz, Graham A.; Cahill, Alison G.; Tuuli, Methodius G.; Obstetrics and Gynecology, School of Medicine
    Importance: Supplemental oxygen is commonly administered to pregnant women at the time of delivery to prevent fetal hypoxia and acidemia. There is mixed evidence on the utility of this practice. Objective: To compare the association of peripartum maternal oxygen administration with room air on umbilical artery (UA) gas measures and neonatal outcomes. Data sources: Ovid MEDLINE, Embase, Scopus, ClinicalTrials.gov, and Cochrane Central Register of Controlled Trials were searched from February 18 to April 3, 2020. Search terms included labor or obstetric delivery and oxygen therapy and fetal blood or blood gas or acid-base imbalance. Study selection: Studies were included if they were randomized clinical trials comparing oxygen with room air at the time of scheduled cesarean delivery or labor in patients with singleton, nonanomalous pregnancies. Studies that did not collect paired umbilical cord gas samples or did not report either UA pH or UA Pao2 results were excluded. Data extraction and synthesis: Data were extracted by 2 independent reviewers. The analysis was stratified by the presence or absence of labor at the time of randomization. Data were pooled using random-effects models. Main outcomes and measures: The primary outcome for this review was UA pH. Secondary outcomes included UA pH less than 7.2, UA Pao2, UA base excess, 1- and 5-minute Apgar scores, and neonatal intensive care unit admission. Results: The meta-analysis included 16 randomized clinical trials (n = 1078 oxygen group and n = 974 room air group). There was significant heterogeneity among the studies (I2 = 49.88%; P = .03). Overall, oxygen administration was associated with no significant difference in UA pH (weighted mean difference, 0.00; 95% CI, -0.01 to 0.01). Oxygen use was associated with an increase in UA Pao2 (weighted mean difference, 2.57 mm Hg; 95% CI, 0.80-4.34 mm Hg) but no significant difference in UA base excess, UA pH less than 7.2, Apgar scores, or neonatal intensive care unit admissions. Umbilical artery pH values remained similar between groups after accounting for the risk of bias, type of oxygen delivery device, and fraction of inspired oxygen. After stratifying by the presence or absence of labor, oxygen administration in women undergoing scheduled cesarean delivery was associated with increased UA Pao2 (weighted mean difference, 2.12 mm Hg; 95% CI, 0.09-4.15 mm Hg) and a reduction in the incidence of UA pH less than 7.2 (relative risk, 0.63; 95% CI, 0.43-0.90), but these changes were not noted among those in labor (Pao2: weighted mean difference, 3.60 mm Hg; 95% CI, -0.30 to 7.49 mm Hg; UA pH<7.2: relative risk, 1.34; 95% CI, 0.58-3.11). Conclusions and relevance: This systematic review and meta-analysis suggests that studies to date showed no association between maternal oxygen and a clinically relevant improvement in UA pH or other neonatal outcomes.