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Archives of Biochemistry and Biophysics Dec 2007The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step...
The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (+/-)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (-)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)- and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase.
Topics: Acyclic Monoterpenes; Diphosphates; Diterpenes; Enzyme Activation; Enzyme Inhibitors; Enzyme Stability; Intramolecular Lyases; Monoterpenes; Polyisoprenyl Phosphates
PubMed: 17949678
DOI: 10.1016/j.abb.2007.09.008 -
ACS Nano Mar 2019Nucleic acid nanostructures have attracted significant interest as potential therapeutic and diagnostic platforms due to their intrinsic biocompatibility and...
Nucleic acid nanostructures have attracted significant interest as potential therapeutic and diagnostic platforms due to their intrinsic biocompatibility and biodegradability, structural and functional diversity, and compatibility with various chemistries for modification and stabilization. Among the fabrication approaches for such structures, the rolling circle techniques have emerged as particularly promising, producing morphologically round, flower-shaped nucleic acid particles: typically hybrid composites of long nucleic acid strands and inorganic magnesium pyrophosphate (MgPPi). These constructs are known to form via anisotropic nucleic acid-driven crystallization in a sequence-independent manner, rendering monodisperse and densely packed RNA or DNA-inorganic composites. However, it still remains to fully explore how flexible polymer-like RNA or DNA strands (acting as biomolecular additives) mediate the crystallization process of MgPPi and affect the structure and properties of the product crystals. To address this, we closely examined nanoscale details to mesoscopic features of MgPPi/DNA hybrid composites fabricated by two approaches, namely rolling circle amplification (RCA)-based in situ synthesis and long synthetic DNA-mediated crystallization. Similar to the DNA constructs fabricated by RCA, the rapid crystallization of MgPPi was retarded on a short-range order when we precipitated the crystals in the presence of presynthesized long DNA, which resulted in effective incorporation of biomolecular additives such as DNA and enzymes. These findings further provide a more feasible way to encapsulate bioactive enzymes within DNA constructs compared to in situ RCA-mediated synthesis, i.e., by not only protecting them from possible denaturation under the reaction conditions but also preventing nonselective association of proteins arising from the RCA reaction mixtures.
Topics: Crystallization; DNA; Diphosphates; Magnesium Compounds; Molecular Structure; Nanostructures; Nucleic Acid Amplification Techniques; Particle Size; Ribonuclease, Pancreatic
PubMed: 30741535
DOI: 10.1021/acsnano.8b06492 -
AIDS (London, England) Dec 2021Tenofovir alafenamide (TAF) preferentially loads peripheral blood mononuclear cells (PBMCs), resulting in higher PBMC tenofovir-diphosphate (TFV-DP) vs. tenofovir... (Randomized Controlled Trial)
Randomized Controlled Trial
Tenofovir-diphosphate in peripheral blood mononuclear cells during low, medium and high adherence to emtricitabine/ tenofovir alafenamide vs. emtricitabine/ tenofovir disoproxil fumarate.
OBJECTIVE
Tenofovir alafenamide (TAF) preferentially loads peripheral blood mononuclear cells (PBMCs), resulting in higher PBMC tenofovir-diphosphate (TFV-DP) vs. tenofovir disoproxil fumarate (TDF). No studies have yet compared TFV-DP in PBMC from lower than daily dosing between prodrugs, which has potential implications for event-driven preexposure prophylaxis and pharmacologic forgiveness.
DESIGN
Two separate randomized, directly observed therapy (DOT) crossover studies (DOT-DBS and TAF-DBS) were conducted to mimic low, medium and high adherence.
METHODS
HIV-negative adults were randomized to two 12-week DOT regimens of 33, 67 or 100% of daily dosing with emtricitabine (F)/TAF 200 mg/25 mg (TAF-DBS) or F/TDF 200 mg/300 mg (DOT-DBS), separated by a 12-week washout. PBMC steady-state concentrations (Css) of TFV-DP and FTC-TP were estimated using nonlinear mixed models and compared between F/TAF and F/TDF.
RESULTS
Thirty-five participants contributed to 33% (n = 23), 67% (n = 23) and 100% (n = 23) of daily F/TAF regimens. Forty-four contributed to 33% (n = 15), 67% (n = 16) and 100% (n = 32) of daily F/TDF regimens. PBMC TFV-DP Css were 7.3 [95% confidence interval (95% CI): 6.4-8.2], 7.1 (5.9-8.2) and 6.7- (4.4-8.9) fold higher (P < 0.0001) following F/TAF vs. F/TDF; 593 vs. 81.7, 407 vs. 57.4, and 215 vs. 32.3 fmol/106 cells, respectively. TFV-DP was 2.6 (2.1-3.1) fold higher with 33% F/TAF vs. 100% F/TDF. Estimated half-lives (95% CI) of TFV-DP in PBMC were 2.9 (1.5-5.5) days for F/TAF and 2.1 (1.5-2.9) days for F/TDF. FTC-TP was similar in both studies (P = 0.119).
CONCLUSION
F/TAF produced 6.7 to 7.3-fold higher TFV-DP in PBMC vs. F/TDF across adherence levels, supporting increased potency and pharmacologic forgiveness with F/TAF in the PBMC compartment.
Topics: Alanine; Anti-HIV Agents; Diphosphates; Emtricitabine; HIV Infections; Humans; Leukocytes, Mononuclear; Tenofovir
PubMed: 34482350
DOI: 10.1097/QAD.0000000000003062 -
British Medical Journal Mar 1979
Topics: Apatites; Arthritis; Crystallization; Diphosphates; Humans
PubMed: 219930
DOI: No ID Found -
Seminars in Arthritis and Rheumatism Jun 2021To explore the lived experience of people with calcium pyrophosphate deposition (CPPD) disease and the impact of this condition on their daily lives.
OBJECTIVE
To explore the lived experience of people with calcium pyrophosphate deposition (CPPD) disease and the impact of this condition on their daily lives.
METHODS
Patients with CPPD and their caregivers were invited to take part in a one-to-one (patient only) or paired (patient and caregiver) semi-structured interview. Interviews covered patients' diagnosis and treatment experiences, and the impact of CPPD on their daily lives. Transcribed interviews were analysed using inductive thematic analysis.
RESULTS
28 patient interviews, six of which included a caregiver, were conducted across five countries. Acute CPP crystal arthritis flares resulted in temporary but profound disability for most patients, disrupting their ability to go about day-to-day activities, and they sought immediate medical attention. CPPD+OA and chronic CPP crystal inflammatory arthritis presented patients with longer term limitations in daily lives. Patients and their caregivers described these disruptions and limitations, which included a reduced ability or inability to complete household and self-care tasks, exercise, socialise, work and drive. They also described how arthritis pain and resulting limitations adversely impacted upon patients' psychological wellbeing. Delays in referral to specialists and diagnostic uncertainty were described by many. Lack of appropriate treatment or access to treatments only upon worsening of symptoms impacted upon the length of time some patients spent in pain and with functional limitations.
CONCLUSION
This study is the first to demonstrate the wide-ranging impact of CPPD, and highlights the need for improved diagnosis, physician training, as well as greater emphasis upon finding targeted therapies to specifically treat CPPD.
Topics: Calcinosis; Calcium Pyrophosphate; Caregivers; Chondrocalcinosis; Diphosphates; Humans
PubMed: 33941385
DOI: 10.1016/j.semarthrit.2021.04.010 -
Trends in Biochemical Sciences Sep 1992The membrane surrounding the central vacuole of plant cells contains an H(+)-translocating ATPase (H(+)-ATPase) and an H(+)-translocating inorganic pyrophosphatase... (Review)
Review
The membrane surrounding the central vacuole of plant cells contains an H(+)-translocating ATPase (H(+)-ATPase) and an H(+)-translocating inorganic pyrophosphatase (H(+)-PPase). Both enzymes are abundant and ubiquitous in plants but the H(+)-PPase is unusual in its exclusive use of inorganic pyrophosphate (PPi) as an energy source. The lack of sequence identity between the vacuolar H(+)-PPase and any other characterized ion pump implies a different evolutionary origin for this translocase. The existence of the vacuolar H(+)-PPase, in conjunction with increasing recognition of PPi as a key metabolite in plant systems, necessitates reconsideration of ATP as the primary energy source for membrane transport in plant cells.
Topics: Amino Acid Sequence; Biological Transport; Cell Membrane; Diphosphates; Molecular Sequence Data; Plants; Proton Pumps; Proton-Translocating ATPases; Vacuoles
PubMed: 1329278
DOI: 10.1016/0968-0004(92)90313-x -
PloS One 2022Knowledge of platelet function in pigs and the effectiveness of antiplatelet therapy is important to ensure proper transferability from animal studies to humans. Our aim...
Knowledge of platelet function in pigs and the effectiveness of antiplatelet therapy is important to ensure proper transferability from animal studies to humans. Our aim was to (1) characterize baseline platelet function of Aachen minipigs using the bedside Multiplate analyzer, (2) compare baseline platelet function with Göttingen minipigs, and (3) characterize platelet inhibition within the first 5 minutes after intravenous administration of acetylsalicylic acid (ASA). We characterized the baseline platelet function and hematological parameters in 9 Aachen minipigs. Historical data of 8 unmedicated Göttingen minipigs were used for comparison of baseline values. Platelet inhibition in Aachen minipigs was tested 1-5 minutes after intravenous administration of 500 mg ASA. Multiplate examinations included the following tests: ASPI test (to assess the effect of ASA), adenosine-diphosphate-test (ADP test) and thrombin receptor activating peptide test (TRAP test). Median values and interquartile range (IQR) of the Multiplate baseline tests in Aachen minipigs were as follows: ASPI: 39 U (IQR = 21-71), ADP: 70 U (IQR = 48-73), and TRAP: 8 U (IQR = 6-9), whereas the values in Göttingen minipigs were as follows: ASPI: 70.5 U (IQR = 60-78), ADP: 51 U (IQR = 45-66), and TRAP: 6.5 U (IQR = 4-8). ASPI values of Göttingen minipigs were significantly higher than those of Aachen minipigs (p = 0.046). Intravenous administration of ASA in Aachen minipigs resulted in significant platelet inhibition after 1 minute, which remained stable over a period of 5 minutes (p≤0.038). Aachen minipigs appeared to have a high variance in arachidonic acid-mediated platelet aggregation. In Aachen minipigs, intravenous ASA administration resulted in immediate platelet inhibition.
Topics: Humans; Swine; Animals; Aspirin; Platelet Aggregation Inhibitors; Swine, Miniature; Arachidonic Acid; Diphosphates; Platelet Function Tests; Platelet Aggregation; Blood Platelets; Adenosine Diphosphate; Receptors, Thrombin; Adenosine
PubMed: 36256639
DOI: 10.1371/journal.pone.0275756 -
Marine Drugs Sep 2022Eukaryotic green microalgae show considerable promise for the sustainable light-driven biosynthesis of high-value fine chemicals, especially terpenoids because of their...
Eukaryotic green microalgae show considerable promise for the sustainable light-driven biosynthesis of high-value fine chemicals, especially terpenoids because of their fast and inexpensive phototrophic growth. Here, the novel isopentenol utilization pathway (IUP) was introduced into to enhance the hemiterpene (isopentenyl pyrophosphate, IPP) titers. Then, diphosphate isomerase (IDI) and limonene synthase (MsLS) were further inserted for limonene production. Transgenic algae showed 8.6-fold increase in IPP compared with the wild type, and 23-fold increase in limonene production compared with a single expressing strain. Following the culture optimization, the highest limonene production reached 117 µg/L, when the strain was cultured in a opt2 medium supplemented with 10 mM isoprenol under a light: dark regimen. This demonstrates that transgenic algae expressing the IUP represent an ideal chassis for the high-value terpenoid production. The IUP will facilitate further the metabolic and enzyme engineering to enhance the terpenoid titers by significantly reducing the number of enzyme steps required for an optimal biosynthesis.
Topics: Chlamydomonas reinhardtii; Diphosphates; Hemiterpenes; Isomerases; Limonene; Metabolic Engineering; Pentanols; Terpenes
PubMed: 36135766
DOI: 10.3390/md20090577 -
Journal of Bone and Mineral Research :... Apr 2023Ectopic calcification is characterized by inappropriate deposition of calcium mineral in nonskeletal connective tissues and can cause significant morbidity and...
Ectopic calcification is characterized by inappropriate deposition of calcium mineral in nonskeletal connective tissues and can cause significant morbidity and mortality, particularly when it affects the cardiovascular system. Identification of the metabolic and genetic determinants of ectopic calcification could help distinguish individuals at the greatest risk of developing these pathological calcifications and could guide development of medical interventions. Inorganic pyrophosphate (PP ) has long been recognized as the most potent endogenous inhibitor of biomineralization. It has been intensively studied as both a marker and a potential therapeutic for ectopic calcification. Decreased extracellular concentrations of PP have been proposed to be a unifying pathophysiological mechanism for disorders of ectopic calcification, both genetic and acquired. However, are reduced plasma concentrations of PP a reliable predictor of ectopic calcification? This perspective article evaluates the literature in favor and against a pathophysiological role of plasma versus tissue PP dysregulation as a determinant of, and as a biomarker for, ectopic calcification. © 2023 American Society for Bone and Mineral Research (ASBMR).
Topics: Humans; Diphosphates; Minerals; Bone and Bones; Calcium, Dietary; Vascular Calcification
PubMed: 36807615
DOI: 10.1002/jbmr.4791 -
Protein Science : a Publication of the... Sep 2022Membrane-bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H pump that transports the H ion generated in coupled pyrophosphate hydrolysis out of...
Membrane-bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H pump that transports the H ion generated in coupled pyrophosphate hydrolysis out of the cytoplasm. Certain bacterial and archaeal mPPases can in parallel transport Na via a hypothetical "billiard-type" mechanism, also involving the hydrolysis-generated proton. Here, we present the functional evidence supporting this coupling mechanism. Rapid-quench and pulse-chase measurements with [ P]pyrophosphate indicated that the chemical step (pyrophosphate hydrolysis) is rate-limiting in mPPase catalysis and is preceded by a fast isomerization of the enzyme-substrate complex. Na , whose binding is a prerequisite for the hydrolysis step, is not required for substrate binding. Replacement of H O with D O decreased the rates of pyrophosphate hydrolysis by both Na - and H -transporting bacterial mPPases, the effect being more significant than with a non-transporting soluble pyrophosphatase. We also show that the Na -pumping mPPase of Thermotoga maritima resembles other dimeric mPPases in demonstrating negative kinetic cooperativity and the requirement for general acid catalysis. The findings point to a crucial role for the hydrolysis-generated proton both in H -pumping and Na -pumping by mPPases.
Topics: Diphosphates; Hydrolysis; Isotopes; Kinetics; Protons; Pyrophosphatases; Sodium; Solvents
PubMed: 36040263
DOI: 10.1002/pro.4394