scholarly journals Candida albicansVMA3Is Necessary for V-ATPase Assembly and Function and Contributes to Secretion and Filamentation

2013 ◽  
Vol 12 (10) ◽  
pp. 1369-1382 ◽  
Author(s):  
Hallie S. Rane ◽  
Stella M. Bernardo ◽  
Summer M. Raines ◽  
Jessica L. Binder ◽  
Karlett J. Parra ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Cellular Response ◽  
Specific Activity ◽  
Calcium Sensitivity ◽  
Vacuolar Membrane ◽  
Cold Sensitivity ◽  
Specific Chemical ◽  
Content Type ◽  
Concanamycin A

ABSTRACTThe vacuolar membrane ATPase (V-ATPase) is a protein complex that utilizes ATP hydrolysis to drive protons from the cytosol into the vacuolar lumen, acidifying the vacuole and modulating several key cellular response systems inSaccharomyces cerevisiae. To study the contribution of V-ATPase to the biology and virulence attributes of the opportunistic fungal pathogenCandida albicans, we created a conditional mutant in whichVMA3was placed under the control of a tetracycline-regulated promoter (tetR-VMA3strain). Repression ofVMA3in the tetR-VMA3strain prevents V-ATPase assembly at the vacuolar membrane and reduces concanamycin A-sensitive ATPase-specific activity and proton transport by more than 90%. Loss ofC. albicansV-ATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. The tetR-VMA3strain also displays abnormal vacuolar morphology, indicative of defective vacuolar membrane fission. The tetR-VMA3strain has impaired aspartyl protease and lipase secretion, as well as attenuated virulence in anin vitromacrophage killing model. Repression ofVMA3suppresses filamentation, and V-ATPase-dependent filamentation defects are not rescued by overexpression ofRIM8,MDS3,EFG1,CST20, orUME6, which encode positive regulators of filamentation. Specific chemical inhibition of Vma3p function also results in defective filamentation. These findings suggest either that V-ATPase functions downstream of these transcriptional regulators or that V-ATPase function during filamentation involves independent mechanisms and alternative signaling pathways. Taken together, these data indicate that V-ATPase activity is a fundamental requirement for several key virulence-associated traits inC. albicans.

scholarly journals A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

2014 ◽  
Vol 59 (1) ◽  
pp. 356-364 ◽  
Author(s):  
Wesley Wu ◽  
Zachary Herrera ◽  
Danny Ebert ◽  
Katie Baska ◽  
Seok H. Cho ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Antimalarial Drug ◽  
Drug Targets ◽  
Specific Activity ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Rescue ◽  
Growth Inhibitory ◽  
Parasite Populations

ABSTRACTThe apicoplast is an essential plastid organelle found inPlasmodiumparasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the “Malaria Box” library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stagePlasmodium falciparumgrowth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations inP. falciparumIspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activityin vitrowith a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action.

scholarly journals Synechocystis KaiC3 Displays Temperature- and KaiB-Dependent ATPase Activity and Is Important for Growth in Darkness

2019 ◽  
Vol 202 (4) ◽  
Author(s):  
Anika Wiegard ◽  
Christin Köbler ◽  
Katsuaki Oyama ◽  
Anja K. Dörrich ◽  
Chihiro Azai ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Circadian Clock ◽  
Atp Hydrolysis ◽  
Synechococcus Elongatus ◽  
Constant Darkness ◽  
Content Type ◽  
Clock Proteins ◽  
Pcc 7942 ◽  
Pcc 6803

ABSTRACT Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies, and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. strain PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of nonstandard KaiC homologs in vitro. KaiC3 displayed ATPase activity lower than that of the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1, and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of ΔkaiAB1C1 and reduced in the absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a cross talk between the KaiC1- and KaiC3-based systems. IMPORTANCE The circadian clock influences the cyanobacterial metabolism, and deeper understanding of its regulation will be important for metabolic optimizations in the context of industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is required. Synechocystis sp. strain PCC 6803 represents a major cyanobacterial model organism and harbors phylogenetically diverged homologs of the clock proteins, which are present in various other noncyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple Synechocystis Kai proteins and characterize enzymatic activities of the nonstandard clock homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness, suggesting its involvement in the regulation of nonphotosynthetic metabolic pathways.

scholarly journals Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

2011 ◽  
Vol 77 (9) ◽  
pp. 2926-2933 ◽  
Author(s):  
Kesaven Bhubalan ◽  
Jo-Ann Chuah ◽  
Fumi Shozui ◽  
Christopher J. Brigham ◽  
Seiichi Taguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Weight Percent ◽  
Pha Synthase ◽  
Content Type ◽  
Highly Active ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

ABSTRACTThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolatedChromobacteriumsp. USM2 (PhaCCs). PhaCCsshowed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. Anin vitroassay of recombinant PhaCCsexpressed inEscherichia colishowed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strainC. necator(307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCswas 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC fromC. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation inEscherichia coliexpressing PhaCCsof up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCsis a naturally occurring, highly active PHA synthase with superior polymerizing ability.

scholarly journals Design of targeted dosage form of ofloxacin+

2006 ◽  
Vol 71 (12) ◽  
pp. 1269-1273 ◽  
Author(s):  
Yar Shahar ◽  
Ahamed Siddiqui ◽  
Ashraf Ali ◽  
E. Manogran
Keyword(s):  
Drug Design ◽  
Specific Activity ◽  
In Vitro Release ◽  
Polymer Backbone ◽  
Specific Chemical ◽  
Site Specific ◽  
Development Of Resistance ◽  
Quinolone Antibiotics ◽  
New Strategy

The targeted pro-drug is a classical pro-drug design often representing a non-specific chemical approach to mask undesirable drug properties, such as limited bioavailability, lack of site specificity, and chemical instability. On the other hand, targeted pro-drug design represents a new strategy for directed and efficient drug delivery. Quinolone antibiotics exert their pharmacological effect by inhibiting the cell wall synthesis of the pathogen. However, development of resistance exists, which instigates for a new higher congener to remain in clinical practice. To overcome this phenomenon and also to produce site-specific activity of the cell walls of the pathogen, ofloxacin is conjugated with a hydroxypropyl methacrylamide polymer backbone moiety. The results of in vitro release studies indicate the possibilities for the development of a new drug for site-specific therapy with an improved t 1/2 of the drug. This novel pro-drugmay have opened a new vista in antibiotic chemotherapy. .

scholarly journals Identity and origin of the ATPase activity associated with neuronal microtubules. II. Identification of a 50,000-dalton polypeptide with ATPase activity similar to F-1 ATPase from mitochondria.

1983 ◽  
Vol 96 (5) ◽  
pp. 1306-1315 ◽  
Author(s):  
D B Murphy ◽  
K T Wallis ◽  
R R Hiebsch
Keyword(s):  
Atpase Activity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Membrane Vesicles ◽  
Cross Reactivity ◽  
Mitochondrial Atpase ◽  
Atpase Inhibitors ◽  
Native Gel ◽  
High Molecular Weight Proteins

We determined that the ATPase activity contained in preparations of neuronal microtubules is associated with a 50,000-dalton polypeptide by four different methods: (a) photoaffinity labeling of the pelletable ATPase fraction with [gamma-32P]-8-azido-ATP; (b) analysis of two-dimensional gels (native gel X SDS slab gel) of an ATPase fraction solubilized by treatment with dichloromethane; (c) ATPase purification by glycerol gradient sedimentation and gel filtration chromatography of a solvent-released ATPase fraction, (d) demonstration of the binding of affinity-purified antibody to the 50-kdalton polypeptide to ATPase activity in vitro. Beginning with preparations of microtubules we have purified the ATPase activity greater than 700-fold and estimate that the purified enzyme has a specific activity of 20 mumol Pi x mg-1 x min-1 and comprises 80-90% of the total ATPase activity associated with neuronal microtubules. With affinity-purified antibody we also demonstrate cross-reactivity to the 50-kdalton subunits of mitochondrial F-1 ATPase and show that the antibody specifically labels mitochondria in PtK-2 cells. Biochemical comparisons of the enzymes reveal similar but not identical subunit composition and sensitivity to mitochondrial ATPase inhibitors. These studies indicate that the principal ATPase activity associated with microtubules is not contained in high molecular weight proteins such as dynein or MAPs and support the hypothesis that the 50-kdalton ATPase is a membrane protein and may be derived from mitochondria or membrane vesicles with F-1-like ATPase activity.

ACE inhibitory activity and bioconversion of isoflavones by Lactobacillus in soymilk supplemented with B‐vitamins

2011 ◽  
Vol 113 (9) ◽  
pp. 1127-1146 ◽  
Author(s):  
Joo‐Ann Ewe ◽  
Wan‐Nadiah Wan‐Abdullah ◽  
Abdul Karim Alias ◽  
Rajeev Bhat ◽  
Min‐Tze Liong
Keyword(s):  
Folic Acid ◽  
Inhibitory Activity ◽  
Specific Activity ◽  
B Vitamins ◽  
Calcium Pantothenate ◽  
Ace Inhibitory Activity ◽  
Content Type ◽  
Bioactive Potential ◽  
Ace Inhibitory

PurposeThe aim of this study is to examine the bioactive properties of lactobacilli‐fermented B‐vitamin soymilk, namely the in‐vitro antihypertensive property and bioconversion of isoflavone glucosides to aglycones.Design/methodology/approachLactobacillus acidophilus BT 1088, L. fermentum BT 8219, L. acidophilus FTDC 8633 and L. gasseri FTDC 8131 were investigated for their bioactive potential and enhanced bioconversion of isoflavones in soymilk supplemented with individual B‐vitamins at a concentration of 1 mg/L.FindingsThe supplementation of thiamine, riboflavin, niacinamide, calcium pantothenate, biotin and folic acid enhanced the ACE‐inhibitory activity of lactobacilli in soymilk accompanied by a lower IC50 value compared to the control (P<0.05). The β‐glucosidase specific activity of lactobacilli was also enhanced on supplementation of B‐vitamins, leading to increased bioconversion of isoflavones in soymilk. The concentration of genistein was decreased, accompanied by an increased concentration of genistein on fermentation in the presence of thiamine, niacinamide, biotin, calcium pantothenate and folic acid. Additionally, the supplementation of niacinamide, calcium pantothenate, biotin and folic acid also led to lower concentrations of malonyl daidzin, indicating increased hydrolysis of malonyl daidzin to daidzin. Results from the present study indicated that the supplementation of B‐vitamins could enhance the bioactive potential and bioconversion of isoflavones in lactobacilli‐fermented soymilk.Originality/valueThis work has shown that the supplementation of B‐vitamins in lactobacilli fermented soymilk has exerted in vitro ACE‐inhibitory activity and increased the accumulation of bioactive isoflavone aglycones. To the authors' knowledge, this is the first evaluation reporting on such aspects.

scholarly journals Hydrogenosome Metabolism Is the Key Target for Antiparasitic Activity of Resveratrol against Trichomonas vaginalis

2013 ◽  
Vol 57 (6) ◽  
pp. 2476-2484 ◽  
Author(s):  
Natalia Mallo ◽  
Jesús Lamas ◽  
José M. Leiro
Keyword(s):  
Gene Expression ◽  
Trichomonas Vaginalis ◽  
Specific Activity ◽  
Treatment Options ◽  
Transmitted Disease ◽  
Protozoan Parasite ◽  
High Dose ◽  
Content Type ◽  
Antiparasitic Drugs

ABSTRACTMetronidazole (MDZ) and related 5-nitroimidazoles are the recommended drugs for treatment of trichomoniasis, a sexually transmitted disease caused by the protozoan parasiteTrichomonas vaginalis. However, novel treatment options are needed, as recent reports have claimed resistance to these drugs inT. vaginalisisolates. In this study, we analyzed for the first time thein vitroeffects of the natural polyphenol resveratrol (RESV) onT. vaginalis. At concentrations of between 25 and 100 μM, RESV inhibited thein vitrogrowth ofT. vaginalistrophozoites; doses of 25 μM exerted a cytostatic effect, and higher doses exerted a cytotoxic effect. At these concentrations, RESV caused inhibition of the specific activity of a 120-kDa [Fe]-hydrogenase (Tvhyd). RESV did not affect Tvhyd gene expression and upregulated pyruvate-ferredoxin oxidoreductase (a hydrogenosomal enzyme) gene expression only at a high dose (100 μM). At doses of 50 to 100 μM, RESV also caused overexpression of heat shock protein 70 (Hsp70), a protective protein found in the hydrogenosome ofT. vaginalis. The results demonstrate the potential of RESV as an antiparasitic treatment for trichomoniasis and suggest that the mechanism of action involves induction of hydrogenosomal dysfunction. In view of the results, we propose hydrogenosomal metabolism as a key target in the design of novel antiparasitic drugs.

scholarly journals A Novel Phosphodiesterase of the GdpP Family Modulates Cyclic di-AMP Levels in Response to Cell Membrane Stress in Daptomycin-Resistant Enterococci

2017 ◽  
Vol 61 (3) ◽  
Author(s):  
Xu Wang ◽  
Milya Davlieva ◽  
Jinnethe Reyes ◽  
Diana Panesso ◽  
Cesar A. Arias ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Stress Responses ◽  
Specific Activity ◽  
Response Regulator ◽  
Physicochemical Characterization ◽  
Membrane Stress ◽  
Signal Molecule ◽  
Content Type ◽  
Bacterial Physiology

ABSTRACT Substitutions in the LiaFSR membrane stress pathway are frequently associated with the emergence of antimicrobial peptide resistance in both Enterococcus faecalis and Enterococcus faecium. Cyclic di-AMP (c-di-AMP) is an important signal molecule that affects many aspects of bacterial physiology, including stress responses. We have previously identified a mutation in a gene (designated yybT) in E. faecalis that was associated with the development of daptomycin resistance, resulting in a change at position 440 (yybT I440S) in the predicted protein. Here, we show that intracellular c-di-AMP signaling is present in enterococci, and on the basis of in vitro physicochemical characterization, we show that E. faecalis yybT encodes a cyclic dinucleotide phosphodiesterase of the GdpP family that exhibits specific activity toward c-di-AMP by hydrolyzing it to 5′pApA. The E. faecalis GdpPI440S substitution reduces c-di-AMP phosphodiesterase activity more than 11-fold, leading to further increases in c-di-AMP levels. Additionally, deletions of liaR (encoding the response regulator of the LiaFSR system) that lead to daptomycin hypersusceptibility in both E. faecalis and E. faecium also resulted in increased c-di-AMP levels, suggesting that changes in the LiaFSR stress response pathway are linked to broader physiological changes. Taken together, our data show that modulation of c-di-AMP pools is strongly associated with antibiotic-induced cell membrane stress responses via changes in GdpP activity or signaling through the LiaFSR system.

scholarly journals PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

2013 ◽  
Vol 80 (2) ◽  
pp. 555-563 ◽  
Author(s):  
Daniel Pfeiffer ◽  
Dieter Jendrossek
Keyword(s):  
De Novo ◽  
Ralstonia Eutropha ◽  
Specific Activity ◽  
High Specific Activity ◽  
Size Exclusion ◽  
Lag Phase ◽  
Granule Formation ◽  
Content Type ◽  
Phb Synthase

ABSTRACTPoly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis inRalstonia eutrophaand other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays ofR. eutrophaPHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-d-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formationin vitroandin vivowas shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection ofde novo-synthesized PHB granules.

scholarly journals BovineStaphylococcus aureusSuperantigens Stimulate the Entire T Cell Repertoire of Cattle

2018 ◽  
Vol 86 (11) ◽  
Author(s):  
Gillian J. Wilson ◽  
Stephen W. Tuffs ◽  
Bryan A. Wee ◽  
Keun Seok Seo ◽  
Nogi Park ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Specific Activity ◽  
Genomic Analysis ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Content Type ◽  
A Genome

ABSTRACTSuperantigens (SAgs) represent a diverse family of bacterial toxins that induce Vβ-specific T cell proliferation associated with an array of important diseases in humans and animals, including mastitis of dairy cows. However, an understanding of the diversity and distribution of SAg genes among bovineStaphylococcus aureusstrains and their role in the pathogenesis of mastitis is lacking. Population genomic analysis of 195 bovineS. aureusisolates representing 57 unique sequence types revealed that strains encode 2 to 13 distinct SAgs and that the majority of isolates contain 5 or more SAg genes. A genome-scale analysis of bovine reference strain RF122 revealed a complement of 11 predicted SAg genes, which were all expressedin vitro. Detection of specific antibodies in convalescent cows suggests expression of 7 of 11 SAgs during naturalS. aureusinfection. We determined the Vβ T cell activation profile for all functional SAgs encoded by RF122, revealing evidence for bovine host-specific activity among the recently identified RF122-encoded SAgs SElY and SElZ. Remarkably, we discovered that some strains have evolved the capacity to stimulate the entire T cell repertoire of cattle through an array of diverse SAgs, suggesting a key role in bovine immune evasion.

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