The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13–22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.
For the first time approved antiretroviral drugs, i.e. protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI), were quantified in dried blood spots (DBS) from HIV/ AIDS patient whole blood samples as the basis for therapeutic drug monitoring (TDM) by a robust simultaneous liquid chromatography/tandem mass spectrometry (LC/MS/MS) method. This study included seven PI (amprenavir, nelfinavir, indinavir, lopinavir, saquinavir, ritonavir, atazanavir) and two NNRTI (nevirapine, efavirenz). LC/MS/MS coupling was realized using a Phenomenex 1 Synergy Max RP LC column (150 Â 2 mm, 4 m) in combination with a tandem mass spectrometer (API 2000, Applied Biosystems/MDS Sciex Concord) operating in positive and negative multiple reaction monitoring (MRM) mode with reserpine as internal standard. DBS samples were punched out and extracted with 50:50 MeOH/0.2 M ZnSO 4 (v/v) as extraction reagent. The method performance data for the drugs in DBS like limits of detection (LOD, 8-70 ng/mL), lower limits of quantification (LLOQ, 41-102 ng/mL), linearity (R 2 , 0.9981-0.9999), linear concentration ranges (41-10.000 ng/mL), accuracies (92-113%), recoveries (62-94%), and ion suppression were investigated and are comparable to data obtained from human plasma, which is the current standard matrix for TDM of PI and NNRTI. In this case, off-line plasma sample preparation was performed by means of simple protein precipitation with 80:20 methanol/0.2 M ZnSO 4 (v/v) as precipitation reagent. Significant correlations between real patient plasma and DBS were obtained for samples containing lopinavir, atazanavir, ritonavir, saquinavir, and efavirenz. DBS preparation as sampling alternative is well suited and practicable for TDM minimizing the high infection risk of HIV/AIDS samples and may facilitate sample mailing.
Soluble guanylyl cyclase (sGC) regulates several important physiological processes by converting GTP into the second-messenger cGMP. sGC has several structural and functional properties in common with adenylyl cyclases (ACs). Recently, we reported that membranous ACs and sGC are potently inhibited by 2',3'-O-(2,4,6-trinitrophenyl)-substituted purine and pyrimidine nucleoside 5'-triphosphates. Using a highly sensitive high-performance liquid chromatography-tandem mass spectrometry method, we report that highly purified recombinant sGC of rat possesses nucleotidyl cyclase activity. As opposed to GTP, ITP, XTP and ATP, the pyrimidine nucleotides UTP and CTP were found to be sGC substrates in the presence of Mn(2+). When Mg(2+) is used, sGC generates cGMP, cAMP, cIMP, and cXMP. In conclusion, soluble "guanylyl" cyclase possesses much broader substrate specificity than previously assumed. Our data have important implications for cyclic nucleotide-mediated signal transduction.
a b s t r a c tPhosphodiesterases (PDEs) capable of degrading cAMP and cGMP are indispensable for the regulation of cyclic nucleotide-mediated signals. The existence of other cyclic nucleotides such as cCMP and cUMP has been discussed controversially in the literature. Despite publications on PDEs hydrolyzing cCMP or cUMP, the molecular identity of such enzymes remained elusive. Recently, we have provided evidence for a role of cCMP as second messenger in vascular relaxation and inhibition of platelet aggregation. Using an HPLC-MS based assay, here, we show that human PDEs belonging to various families hydrolyze not only cAMP and cGMP but also other cyclic nucleotides.
Cyclic dinucleotides such as bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) and bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) represent important second messengers in bacteria. Although their synthesis has not been described in plants so far, they may be involved in the regulation of bacterial phytopathogen-plant interactions as well as rhizobium plant symbiosis. Here, we describe a sensitive and specific quantification method for c-di-AMP and c-di-GMP by HPLC-coupled tandem mass spectrometry. Additional linear dinucleotide metabolites and mononucleotides, as well as cyclic mononucleotides, can be simultaneously determined by this method.
SummaryBackground and objectives The fixed antibacterial combination of ampicillin and sulbactam is frequently used for various infections. Intact kidneys eliminate approximately 71% of ampicillin and 78% of sulbactam. Patients on thrice-weekly low-flux hemodialysis exhibit an ampicillin t 1/2 of 2.3 hours on and 17.4 hours off dialysis. Despite its frequent use in intensive care units, there are no available dosing recommendations for patients with AKI undergoing renal replacement therapy. The aims of this study were to evaluate the pharmacokinetics of ampicillin/ sulbactam in critically ill patients with AKI undergoing extended dialysis (ED) and to establish a dosing recommendation for this treatment method.Design, setting, participants, & measurements Twelve critically ill patients with anuric AKI being treated with ED were enrolled in a prospective, open-label, observational pharmacokinetic study. Pharmacokinetics after a single dose of ampicillin/sulbactam (2 g/1 g) was obtained in 12 patients. Multiple-dose pharmacokinetics after 4 days of twice-daily ampicillin/sulbactam (2 g/1 g) was obtained in three patients. ResultsThe mean dialyzer clearance for ampicillin/sulbactam was 80.167.7/83.3612.1 ml/min. The t 1/2 of ampicillin and sulbactam in patients with AKI undergoing ED were 2.860.8 hours and 3.561.5 hours, respectively. There was no significant accumulation using a twice-daily dosage of 2 g/1 g ampicillin/sulbactam.Conclusions Our data suggest that in patients treated with ED using a high-flux dialyzer (polysulphone, 1.3 m 2 ; blood and dialysate flow, 160 ml/min; treatment time, 480 minutes), a twice-daily dosing schedule of at least 2 g/1 g ampicillin/sulbactam, with one dose given after ED, should be used to avoid underdosing.
Purines are a class of small organic molecules that are essential for all cells. They play critical roles in neuronal differentiation and function. Their importance is highlighted by several inherited disorders of purine metabolism, such as the Lesch-Nyhan disease, which is caused by a deficiency of the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt). Despite the known importance of purines in the nervous system, knowledge regarding their metabolism in neurons is limited. In the current studies, purine pools and their metabolism were examined in rat PC6-3 cells, a PC12 pheochromocytoma subclone that undergoes robust differentiation with nerve growth factor. The results were compared with five new independent PC6-3 subclones with defective purine recycling due to different mutations affecting HGprt enzyme activity. The results demonstrate an increase in most purines and in energy state following neuronal differentiation, as well as specific abnormalities when purine recycling is lost. The loss of HGprt-mediated purine recycling also is associated with significant loss of dopamine and related metabolites in the mutant PC6-3 lines, suggesting an important connection between purine and dopamine pathways. These results provide insights into how purine pools and metabolism change with neuronal differentiation, and how specific enzyme defects may cause neuronal dysfunction.
Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.