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Biochemistry Jun 2015Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three...
Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1'-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1'-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO₂ from the carboxyphosphate intermediate to biotin.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Bacterial Proteins; Bicarbonates; Biocatalysis; Biotin; Carbon-Nitrogen Ligases; Catalytic Domain; Crystallography, X-Ray; Databases, Protein; Foscarnet; Haemophilus influenzae; Models, Molecular; Molecular Conformation; Organophosphorus Compounds; Phosphates; Protein Conformation; Protein Subunits
PubMed: 26020841
DOI: 10.1021/acs.biochem.5b00340 -
Physiological Research Aug 2020Lithium is mainly excreted into urine, and a large fraction of lithium filtered through glomeruli is reabsorbed in the proximal tubule. However, the mechanisms...
Lithium is mainly excreted into urine, and a large fraction of lithium filtered through glomeruli is reabsorbed in the proximal tubule. However, the mechanisms responsible for lithium reabsorption remain unclear. We previously reported that the reabsorption of lithium was biphasic in rats, and that foscarnet inhibited lithium reabsorption with a high affinity type. We herein evaluated the effects of acetazolamide and foscarnet on the renal excretion of lithium in rats treated with lithium chloride at 2 doses. In rats intravenously injected with a bolus of 25 mg/kg lithium chloride, acetazolamide facilitated the urinary excretion of lithium, and increased the fractional excretion of lithium from 0.446 to 0.953, near the theoretically maximum value. At a dose of 2.5 mg/kg lithium chloride, the fractional excretion of lithium was 0.241 in control rats, 0.420 in rats administered acetazolamide, and 0.976 in rats administered acetazolamide and foscarnet. These results showed the potent inhibition of lithium reabsorption by acetazolamide and foscarnet in rats. And, it was exhibited that the effects of acetazolamide on lithium reabsorption differed with the dosages of lithium administered.
Topics: Acetazolamide; Animals; Antiviral Agents; Disease Models, Animal; Diuretics; Drug Interactions; Foscarnet; Kidney Tubules, Proximal; Lithium Chloride; Male; Rats; Rats, Wistar; Renal Reabsorption
PubMed: 32584131
DOI: 10.33549/physiolres.934285 -
Biochimica Et Biophysica Acta.... Sep 2019Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter...
Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of K = 1.387 ± 0.1674 mM Pi and V = 198.6 ± 10.23 Pi × h × mg protein hence reflecting a low affinity Pi transport. H-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H-dependent Pi transport was five-fold higher than the Na-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.
Topics: Breast Neoplasms; Cadherins; Cell Adhesion; Cell Line; Cell Proliferation; Down-Regulation; Female; Humans; Hydrogen-Ion Concentration; Ion Transport; Kinetics; Phosphate Transport Proteins; Phosphates; Phosphonoacetic Acid; Sodium-Phosphate Cotransporter Proteins, Type IIb; Tumor Microenvironment; Up-Regulation
PubMed: 31034992
DOI: 10.1016/j.bbadis.2019.04.015 -
Structure (London, England : 1993) Jul 2016CAD, the multienzymatic protein that initiates and controls de novo synthesis of pyrimidines in animals, associates through its aspartate transcarbamoylase (ATCase)...
CAD, the multienzymatic protein that initiates and controls de novo synthesis of pyrimidines in animals, associates through its aspartate transcarbamoylase (ATCase) domain into particles of 1.5 MDa. Despite numerous structures of prokaryotic ATCases, we lack structural information on the ATCase domain of CAD. Here, we report the structure and functional characterization of human ATCase, confirming the overall similarity with bacterial homologs. Unexpectedly, human ATCase exhibits cooperativity effects that reduce the affinity for the anti-tumoral drug PALA. Combining structural, mutagenic, and biochemical analysis, we identified key elements for the necessary regulation and transmission of conformational changes leading to cooperativity between subunits. Mutation of one of these elements, R2024, was recently found to cause the first non-lethal CAD deficit. We reproduced this mutation in human ATCase and measured its effect, demonstrating that this arginine is part of a molecular switch that regulates the equilibrium between low- and high-affinity states for the ligands.
Topics: Antineoplastic Agents; Aspartate Carbamoyltransferase; Aspartic Acid; Catalytic Domain; Enzyme Inhibitors; Humans; Phosphonoacetic Acid
PubMed: 27265852
DOI: 10.1016/j.str.2016.05.001 -
Medicine Feb 2022Cytomegalovirus (CMV) disease is relatively uncommon in nontransplant hematological patients. Moreover, cutaneous manifestations of CMV diseases have scarcely been...
RATIONALE
Cytomegalovirus (CMV) disease is relatively uncommon in nontransplant hematological patients. Moreover, cutaneous manifestations of CMV diseases have scarcely been reported and are probably under-recognized.
PATIENT CONCERNS
We describe a patient with large B-cell lymphoma who developed a band-form, erythematous lesion over his left abdomen soon after the second course of rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone chemotherapy.
DIAGNOSES
The lesion was initially mistaken for bacterial cellulitis or herpes zoster and was histologically confirmed as cutaneous CMV infection. Subsequent work-up also detected CMV viremia and the presence of CMV meningoencephalitis.
INTERVENTIONS
The patient was treated with ganciclovir plus CMV immune globulin followed by foscarnet.
OUTCOMES
Although the patient's cutaneous lesion resolved, his cognitive impairment did not recover, and he developed a fatal multi-organ failure 1 month later.
LESSONS
Cutaneous CMV disease can herald multisystem involvement and an unfavorable prognosis in immunocompromised hosts. It should be ruled out with biopsy in patients with hematological malignancy who have cutaneous lesions refractory to antibacterial therapy.
Topics: Antiviral Agents; Cytomegalovirus Infections; Fatal Outcome; Foscarnet; Ganciclovir; Hematologic Neoplasms; Humans; Immunocompromised Host; Male; Skin Diseases
PubMed: 35119018
DOI: 10.1097/MD.0000000000028721 -
Chemical & Pharmaceutical Bulletin Jan 2024The spectrum of P-NMR is fundamentally simpler than that of H-NMR; consequently identifying the target signal(s) for quantitation is simpler using quantitative P-NMR...
The spectrum of P-NMR is fundamentally simpler than that of H-NMR; consequently identifying the target signal(s) for quantitation is simpler using quantitative P-NMR (P-qNMR) than using quantitative H-NMR (H-qNMR), which has been already established as an absolute determination method. We have previously reported a P-qNMR method for the absolute determination of cyclophosphamide hydrate and sofosbuvir as water-soluble and water-insoluble organophosphorus compounds, respectively. This study introduces the purity determination of brigatinib (BR), an organophosphorus compound with limited water solubility, using P-qNMR at multiple laboratories. Phosphonoacetic acid (PAA) and 1,4-BTMSB-d were selected as the reference standards (RSs) for P-qNMR and H-qNMR, respectively. The qNMR solvents were chosen based on the solubilities of BR and the RSs for qNMR. CDOH was selected as the solvent for P-qNMR measurements to prevent the influence of deuterium exchange caused by the presence of exchangeable intramolecular protons of BR and PAA on the quantitative values, while CDOD was the solvent of choice for the H-qNMR measurements to prevent the influence of water signals and the exchangeable intramolecular protons of BR and PAA. The mean purity of BR determined by P-qNMR was 97.94 ± 0.69%, which was in agreement with that determined by H-qNMR (97.26 ± 0.71%), thus indicating the feasibility of purity determination of BR by P-qNMR. Therefore, the findings of this study may provide an effective method that is simpler than conventional H-qNMR for the determination of organophosphorus compounds.
Topics: Protons; Organophosphorus Compounds; Reference Standards; Water; Solvents
PubMed: 37899177
DOI: 10.1248/cpb.c23-00635 -
Journal of Neurovirology Apr 2017Acyclovir resistance is rarely seen in herpes simplex virus (HSV) type I encephalitis. Prevalence rates vary between 0.5 % in immunocompetent patients (Christophers et...
Acyclovir resistance is rarely seen in herpes simplex virus (HSV) type I encephalitis. Prevalence rates vary between 0.5 % in immunocompetent patients (Christophers et al. 1998; Fife et al. 1994) and 3.5-10 % in immunocompromised patients (Stranska et al. 2005). We report a 45-year-old, immunocompetent (negative HIV antigen/antibody testing), female patient, without previous illness who developed-after a febrile prodromal stage-aphasia and psychomotor slowing. Cerebral magnetic resonance imaging (cMRI) showed right temporal and insular T2-hyperintense lesions with spreading to the contralateral temporal lobe. Cerebrospinal fluid (CSF) analysis yielded lymphocytic pleocytosis and elevated protein level. Polymerase chain reaction testing for HSV type I showed a positive result in repeat lumbar puncture. HSV type I encephalitis was diagnosed and intravenous acyclovir treatment was initiated (750 mg t.i.d.). Acyclovir treatment was intensified to 1000 mg t.i.d., due to clinical deterioration, ongoing pleocytosis and progression on cMRI 5 days after initiation of antiviral therapy. In parallel, acyclovir resistance testing showed mutation of thymidine kinase gene at position A156V prompting foscarnet therapy (60 mg t.i.d.). Patient's condition improved dramatically over 2 weeks. Acyclovir resistance is rare but should be considered in case of clinical worsening of patient's condition. To our knowledge, this is the first report of acyclovir resistance in HSV type I encephalitis of an immunocompetent and previously healthy patient in Austria.
Topics: Acyclovir; Antiviral Agents; Disease Progression; Drug Resistance, Viral; Drug Substitution; Encephalitis, Herpes Simplex; Female; Foscarnet; Herpes Simplex; Herpesvirus 1, Human; Humans; Leukocytosis; Magnetic Resonance Imaging; Middle Aged; Temporal Lobe
PubMed: 27787806
DOI: 10.1007/s13365-016-0489-5 -
Transplantation Oct 2016Antiviral-resistant or refractory cytomegalovirus (CMV) infection is challenging, and salvage therapies, foscarnet, and cidofovir, have significant toxicities. Several...
BACKGROUND
Antiviral-resistant or refractory cytomegalovirus (CMV) infection is challenging, and salvage therapies, foscarnet, and cidofovir, have significant toxicities. Several investigational anti-CMV agents are under development, but more information is needed on outcomes of current treatments to facilitate clinical trial design for new drugs.
METHODS
Records of solid organ transplant (SOT) and hematopoietic cell transplant (HCT) recipients at a single center over a 10-year period were reviewed retrospectively to characterize those who had received foscarnet treatment for ganciclovir-resistant or refractory CMV infection. Data were collected on virologic responses, mortality, and nephrotoxicity.
RESULTS
Of 39 patients (22 SOT, 17 HCT), 15 had documented ganciclovir resistance mutations and 11 (28%) of 39 had tissue-invasive CMV. Median duration of foscarnet was 32 days. Virologic failure occurred in 13 (33%) of 39 and relapses of viremia occurred in 31%. Mortality was 12 (31%) of 39 and was higher in HCT than SOT (P = 0.001), although ganciclovir resistance was more common in SOT (P = 0.003). Doses of ganciclovir or valganciclovir were low in 10 (26%) of 39 at some time before switching to foscarnet. Renal dysfunction occurred in 20 (51%) of 39 by end of treatment and in 7 (28%) of 25 after 6 months.
CONCLUSIONS
Outcomes of existing treatment for ganciclovir-resistant or refractory CMV are suboptimal, in terms of virologic clearance, renal dysfunction, and mortality. These data should provide background information for future clinical trials of newer antiviral agents.
Topics: Adolescent; Adult; Aged; Antiviral Agents; Child; Cytomegalovirus Infections; Drug Resistance, Viral; Female; Foscarnet; Ganciclovir; Hematopoietic Stem Cell Transplantation; Humans; Male; Middle Aged; Retrospective Studies; Transplant Recipients
PubMed: 27495775
DOI: 10.1097/TP.0000000000001418 -
Biology of Blood and Marrow... Jun 2018Cord blood transplantation (CBT) is a distinct risk factor for human herpesvirus-6 (HHV-6) reactivation and HHV-6 encephalitis. In a prospective multicenter trial we... (Clinical Trial)
Clinical Trial
Effects of Prophylactic Foscarnet on Human Herpesvirus-6 Reactivation and Encephalitis in Cord Blood Transplant Recipients: A Prospective Multicenter Trial with an Historical Control Group.
Cord blood transplantation (CBT) is a distinct risk factor for human herpesvirus-6 (HHV-6) reactivation and HHV-6 encephalitis. In a prospective multicenter trial we investigated the effects of prophylactic foscarnet (90 mg/kg i.v. infusion from days 7 to 27 after CBT) on the occurrence of HHV-6 reactivation, HHV-6 encephalitis, and acute graft-versus-host disease (aGVHD) in CBT recipients. Between 2014 and 2016, 57 patients were included in a foscarnet-prophylaxis group. Outcomes were compared with an historical control group who received CBT between 2010 and 2014 (standard-treatment group, n = 63). The cumulative incidence of high-level HHV-6 reactivation, defined as plasma HHV-6 DNA ≥ 10 copies/mL, at 60 days after CBT was significantly lower in the foscarnet-prophylaxis group than in the standard-treatment group (18.3% versus 57.3%, P < .001). Multivariate analysis revealed that myeloablative preconditioning and standard treatment were significant risk factors for high-level HHV-6 reactivation. The cumulative incidence of HHV-6 encephalitis at 60 days after CBT was not different between the groups (foscarnet-prophylaxis group, 12.4%; standard-treatment group, 4.9%; P = .14). The cumulative incidences of grades II to IV and grades III to IV aGVHD at 60 days after CBT were not different between the groups (grades II to IV aGVHD: foscarnet-prophylaxis group, 42.0%; standard-treatment group, 40.5%; P = .96; grades III to IV aGVHD: foscarnet-prophylaxis group, 14.5%; standard-treatment group, 14.5%; P = 1.00). In the setting of this study foscarnet significantly suppressed systemic HHV-6 reactivation in CBT recipients but failed to prevent the development of HHV-6 encephalitis. Suppression of HHV-6 reactivation by foscarnet did not show any effects against the incidence of aGVHD.
Topics: Adolescent; Adult; Aged; Antiviral Agents; DNA, Viral; Encephalitis, Viral; Female; Fetal Blood; Foscarnet; Graft vs Host Disease; Herpesvirus 6, Human; Historically Controlled Study; Humans; Middle Aged; Myeloablative Agonists; Premedication; Prospective Studies; Risk Factors; Treatment Outcome; Virus Activation; Young Adult
PubMed: 29454651
DOI: 10.1016/j.bbmt.2018.02.008 -
Journal of Vascular Surgery Jul 2015Hyperphosphatemia-induced endothelial dysfunction has been shown to play a pathogenic role in the development of atherosclerosis in chronic kidney disease (CKD) through...
OBJECTIVE
Hyperphosphatemia-induced endothelial dysfunction has been shown to play a pathogenic role in the development of atherosclerosis in chronic kidney disease (CKD) through unclear mechanisms. Emerging evidence indicates that autophagy is involved in the maintenance of normal cardiovascular function. However, it is unclear whether autophagy participates in the molecular mechanism underlying high phosphate (Pi)-induced endothelial dysfunction.
METHODS
The autophagy activity was determined by the immunofluorescence staining of the expression of endothelial microtubule-associated protein 1 light chain 3 (LC3) in the 5/6 nephrectomy rat model of CKD and sham-operated control rats. The LC3-II/LC3-I ratio and the activation of the Akt/mammalian target of rapamycin (mTOR) signaling pathway were determined in cultured human microvascular endothelial cell (HMEC-1) endothelial cells that were exposed to a high concentration of Pi with or without the Pi influx blocker phosphonoformic acid, the autophagy inhibitor 3-methyladenine, and the autophagy inducer rapamycin. The impacts of autophagy on Pi-induced apoptotic damage were assessed by flow cytometry.
RESULTS
The in vivo rat model of CKD revealed that hyperphosphatemia is associated with increased endothelial LC3 staining. The exposure of HMEC-1 cells to high Pi induced both dose-dependent and time-dependent increases in the LC3-II/LC3-I expression ratio accompanied by the inhibition of the Akt/mTOR signaling pathway. In HMEC-1 cells, high Pi-induced autophagy and the inhibition of Akt/mTOR signaling were reversed by phosphonoformic acid through the blockage of Pi influx. Apoptosis, characterized by the levels of cleaved caspase 3 and poly(ADP-ribose) polymerase, along with autophagy was induced by high Pi, and the inhibition of autophagy by 3-methyladenine significantly aggravated high Pi-induced apoptosis. The flow cytometry results confirmed that the blockage of autophagy promoted the apoptosis of endothelial cells.
CONCLUSIONS
Hyperphosphatemia induces endothelial autophagy, possibly through the inhibition of the Akt/mTOR signaling pathway, which may play a protective role against high Pi-induced apoptosis.
Topics: Adenine; Animals; Autophagy; Cell Line; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Activation; Foscarnet; Humans; Hyperphosphatemia; Male; Microtubule-Associated Proteins; Phosphates; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats, Wistar; Renal Insufficiency, Chronic; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Time Factors; Transfection
PubMed: 24797554
DOI: 10.1016/j.jvs.2014.02.040