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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 -
Journal of Virology Sep 1978The timing of some of the molecular events that are required for cell fusion was investigated. Cell fusion was produced by a mutant of herpes simplex virus type 1 that...
The timing of some of the molecular events that are required for cell fusion was investigated. Cell fusion was produced by a mutant of herpes simplex virus type 1 that causes extensive cell fusion during infection. The timing of molecular events required for fusion was established by the use of blocking agents. Phosphonoacetic acid blocks viral DNA synthesis; actinomycin D blocks RNA synthesis; cycloheximide blocks protein synthesis; 2-deoxyglucose blocks glycosylation of glycoproteins; high temperature, NH(4)Cl, and adamantanone block unknown steps required for cell fusion. For cells infected at a low multiplicity of infection, phosphonoacetic acid decreased the rate but not the final amount of fusion, but at a multiplicity of infection of 10 it had no effect on the rate of cell fusion. RNA synthesis was required for fusion until 4 h after infection, protein synthesis until 5.5 h after infection, and glycosylation until 7 h after infection. The temperature-dependent step occurred before 6 h after infection, whereas NH(4)Cl and adamantanone acted at steps that occurred until 8 h after infection. Cycloheximide, temperature, NH(4)Cl, and adamantanone acted reversibly; actinomycin D and 2-deoxyglucose acted irreversibly. The same order of action of the inhibitors was also determined by using pairs of inhibitors sequentially. These experiments also indicated that the fusion factor was not an alpha-polypeptide. Virus growth and cell fusion were both found to be highly dependent on temperature in the range of 30 to 40 degrees C. Wild-type infections are apparently characterized by the presence of a fusion factor and a fusion inhibitor. The fusion-blocking agents were added to wild-type-infected cells under a variety of conditions in an attempt to selectively block the production of the fusion inhibitor molecule and thereby cause extensive cell fusion. However, fusion was not observed in any of these experiments.
Topics: Adamantane; Ammonium Chloride; Cell Fusion; Cell Line; Cycloheximide; DNA Replication; DNA, Viral; Dactinomycin; Deoxyglucose; Phosphonoacetic Acid; Simplexvirus; Temperature; Virus Replication
PubMed: 212579
DOI: 10.1128/JVI.27.3.505-512.1978 -
Antimicrobial Agents and Chemotherapy Sep 1995Hypocalcemia and an increase in creatinine level are the most important serious effects associated with foscarnet (PFA) therapy. In an animal model, we have explored the...
Hypocalcemia and an increase in creatinine level are the most important serious effects associated with foscarnet (PFA) therapy. In an animal model, we have explored the potential protective role of liposome-encapsulated foscarnet (LE-PFA) on these metabolic abnormalities. PFA administered as one bolus injection (0.5 or 1.0 g/kg) caused significant rapid decreases (approximately 20%) in the levels of calcium and phosphorus in serum within a few minutes and up to 30 min after injection. LE-PFA did not induce any of these changes, while peak levels in serum and the half-life of this formulation were much higher than those of the free drug. PFA administered for 2 weeks (340 or 500 mg/kg/day) resulted in no changes in creatinine or blood urea nitrogen levels in serum at the low-dosage level, but at the higher-dosage level, the creatinine level in serum increased by day 5 posttreatment. Furthermore, there was no increase in the creatinine or blood urea nitrogen level after 2 weeks of treatment with LE-PFA at a dosage of 35 mg/kg/day. When the pharmacokinetics of both free PFA and LE-PFA were compared, the plasma half-life of the encapsulated drug was approximately four times longer than that of the free drug. In addition, the systemic clearance of LE-PFA was approximately one-fifth of that of the free drug. In conclusion, free PFA causes hypocalcemia and hypophosphatemia and increases the creatinine level in serum, whereas the LE form of this drug seems to protect against the abnormal changes in calcium and phosphorus levels caused by the free drug. By preventing hypocalcemia and increasing its half-life, LE-PFA can be used at lower doses and at longer intervals. Clinical investigations of these formulations may be worthwhile.
Topics: Animals; Antiviral Agents; Calcium; Dose-Response Relationship, Drug; Drug Carriers; Female; Foscarnet; Half-Life; Hypocalcemia; Liposomes; Mice; Mice, Inbred C57BL; Phosphorus
PubMed: 8540701
DOI: 10.1128/AAC.39.9.1973 -
Journal of Virology Aug 2004Infection of endothelial cells with human herpesvirus 8 (HHV-8) is an essential event in the development of Kaposi's sarcoma. When primary microvascular endothelial...
Infection of endothelial cells with human herpesvirus 8 (HHV-8) is an essential event in the development of Kaposi's sarcoma. When primary microvascular endothelial cells (MECs) were infected with HHV-8 at a low multiplicity of infection, considerable latent replication of HHV-8 occurred, leading to a time-dependent increase in the percentage of virus-infected cells that was accompanied by cellular spindling and growth to a high density with loss of contact inhibition. Only a low percentage of MECs supported lytic replication of HHV-8 and produced infectious virus. Phosphonoformic acid blocked production of infectious virus but did not inhibit the rapid expansion of latently infected MECs. Pretreatment of MECs with alpha interferon (IFN-alpha) prior to infection effectively reduced HHV-8 viral gene expression, latent replication, and production of infectious virus. High levels of the double-stranded RNA activated protein kinase (PKR) were expressed in HHV-8-infected cells, and incubation with IFN-alpha increased PKR expression more in virus-infected cells than in uninfected cells. MECs that were immortalized with simian virus 40 large-T antigen differed from nonimmortalized MECs in their response to infection with HHV-8 and demonstrated that cells with elevated levels of expression of antiviral transcripts expressed viral transcripts at reduced levels. These studies demonstrate that MECs respond to HHV-8 with enhanced expression of cellular antiviral genes and that augmentation of innate antiviral defenses with IFN-alpha is a more effective strategy than inhibition of viral lytic replication to protect MECs from infection with HHV-8 and to restrict proliferation of virus-infected MECs.
Topics: Antigens, Viral; Cell Division; Cells, Cultured; Endothelial Cells; Foscarnet; Herpesvirus 8, Human; Humans; Interferon-alpha; Nuclear Proteins; Nucleic Acid Synthesis Inhibitors; Virus Replication; eIF-2 Kinase
PubMed: 15254208
DOI: 10.1128/JVI.78.15.8359-8371.2004 -
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 -
Chemistry & Biology Oct 2011Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to...
Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 Å resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases but with notable differences, such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional cocrystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently bound transition state mimic provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily.
Topics: Acetates; Alkaline Phosphatase; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Enzyme Inhibitors; Hydrolysis; Metals; Models, Molecular; Phosphonoacetic Acid; Protein Conformation; Sinorhizobium meliloti; Substrate Specificity
PubMed: 22035792
DOI: 10.1016/j.chembiol.2011.07.019 -
Journal of Molecular Biology Jul 2010Structure-based protein sequence alignments of family B DNA polymerases revealed a conserved motif that is formed from interacting residues between loops from the...
Structure-based protein sequence alignments of family B DNA polymerases revealed a conserved motif that is formed from interacting residues between loops from the N-terminal and palm domains and between the N-terminal loop and a conserved proline residue. The importance of the motif for function of the bacteriophage T4 DNA polymerase was revealed by suppressor analysis. T4 DNA polymerases that form weak replicating complexes cannot replicate DNA when the dGTP pool is reduced. The conditional lethality provides the means to identify amino acid substitutions that restore replication activity under low-dGTP conditions either by correcting the defect produced by the first amino acid substitution or by generally increasing the stability of polymerase complexes; the second type are global suppressors that can effectively counter the reduced stability caused by a variety of amino acid substitutions. Some amino acid substitutions that increase the stability of polymerase complexes produce a new phenotype-sensitivity to the antiviral drug phosphonoacetic acid. Amino acid substitutions that confer decreased ability to replicate DNA under low-dGTP conditions or drug sensitivity were identified in the new motif, which suggests that the motif functions in regulating the stability of polymerase complexes. Additional suppressor analyses revealed an apparent network of interactions that link the new motif to the fingers domain and to two patches of conserved residues that bind DNA. The collection of mutant T4 DNA polymerases provides a foundation for future biochemical studies to determine how DNA polymerases remain stably associated with DNA while waiting for the next available dNTP, how DNA polymerases translocate, and the biochemical basis for sensitivity to antiviral drugs.
Topics: Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Antiviral Agents; Bacteriophage T4; DNA Mutational Analysis; DNA-Directed DNA Polymerase; Models, Molecular; Molecular Sequence Data; Phosphonoacetic Acid; Protein Stability; Protein Structure, Tertiary; Sequence Alignment; Suppression, Genetic; Viral Plaque Assay; Viral Proteins
PubMed: 20493878
DOI: 10.1016/j.jmb.2010.05.030 -
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 -
Journal of Virology Jun 1980Phenotypic and genetic properties of 12 markers in structural and regulatory functions of herpes simplex virus type 1 were characterized, and their recombination and...
Phenotypic and genetic properties of 12 markers in structural and regulatory functions of herpes simplex virus type 1 were characterized, and their recombination and segregation behavior was investigated and interpreted with reference to available information on their physical locations. The markers were: (i) ts markers in a structural glycoprotein (tsB5) and in alpha (immediate early; tsLB2, tsc75) or beta (early, delayed early; tsB1) functions with regulatory effects; together with (ii) plaque morphology (syn), phosphonoacetate resistance (Pr), and thymidine kinase (TK) phenotypes; and (iii) electrophoretically distinct variants of glycosylated (glycoprotein C, gpC; ICP10) and non-glycosylated [VP(13-14), VP23] structural and nonstructural [ICP(47-48)] polypeptides. Mean two-factor recombination frequencies ranged from 2% (for noncomplementing mutants tsLB2 and tsc75) to 35 to 40% (for unlinked markers) and were influenced by the relative contributions of parental viruses to the mixed infection. Even with control of this variable, standard deviations of mean measures of recombination frequency ranged from a minimum of 14% (with n greater than or equal to 10) to 65% (with n = 3) of mean values; no recombination frequencies higher than 55% were observed. Differences in mean two-factor recombination frequencies between a small number of loosely linked markers were, therefore, not reliable measures of real differences in linkage. Measurements of the segregation of unselected markers among recombinant progeny were, therefore, used as measures of linkage. These experiments (i) established a linkage group for markers in the long unique region of the genome additional to, but consistent with, existing physical data, i.e., TK-syn-tsB5-(tsB1.Pr)-[gpC.VP(13-14)]; (II) identified markers, e.g., ICP(47-48), linked to regulatory mutations (tsLB2, tsc75) in redundant DNA sequences; and (iii) used the segregation of these regulatory mutations and linked markers among unselected progeny to demonstrate the linkage groups: Pr-syn-TK-tsc75-ICP(47-48), [VP(13-14).gpC]-Pr-syn-TK, and TK-tsc75-[VP(13-14).gpC]. These results were most simply explained if bi- or intermolecular recombination occurred between circular molecules or molecules catenated "head-to-tail" and were incompatible with intermolecular recombination as the mechanism of isomerization of herpes simplex virus DNA.
Topics: Genes; Genes, Regulator; Genes, Viral; Genetic Complementation Test; Genetic Linkage; Genetic Markers; Phosphonoacetic Acid; Recombination, Genetic; Simplexvirus; Thymidine Kinase; Viral Plaque Assay; Viral Proteins
PubMed: 6247508
DOI: 10.1128/JVI.34.3.716-742.1980 -
Cellular Physiology and Biochemistry :... 2007In mineralising tissues such as growth plate cartilage extracellular organelles derived from the chondrocyte membrane are present. These matrix vesicles (MV) possess...
In mineralising tissues such as growth plate cartilage extracellular organelles derived from the chondrocyte membrane are present. These matrix vesicles (MV) possess membrane transporters that accumulate Ca(2+) and inorganic phosphate (P(i)), and initiate the formation of hydroxyapatite crystals. MV are also present in articular cartilage, and hydroxyapatite crystals are believed to promote cartilage degradation in osteoarthritic joints. In the present study, P(i) transport pathways in isolated bovine articular chondrocytes have been characterised. P(i) uptake was temperature-sensitive and could be resolved into Na(+)-dependent and Na(+)-independent components. The Na(+)-dependent component saturated at high concentrations of extracellular P(i), with a K(m) for P(i) of 0.17 mM. In solutions lacking Na(+), uptake did not fully saturate, implying that under these conditions carrier-mediated uptake is supplemented by a diffusive pathway. Both Na(+)-dependent and Na(+)-independent components were sensitive to the P(i) transport inhibitors phosphonoacetate and arsenate, although a fraction of Na(+)-independent P(i) uptake was resistant to these anions. Total P(i) uptake was optimal at pH 7.4, and reduced as pH was made more acidic or more alkaline, an effect that represented reduced Na(+)-dependent influx. RT-PCR analysis confirmed that two members of the NaPi III family, Pit-1 and Pit-2, are expressed, but that NaPi II transporters are not.
Topics: Animals; Arsenates; Base Sequence; Biological Transport, Active; Cartilage, Articular; Cattle; Chondrocytes; DNA Primers; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Male; Phosphate Transport Proteins; Phosphates; Phosphonoacetic Acid; RNA; Sodium; Sodium-Phosphate Cotransporter Proteins, Type III; Sodium-Phosphate Cotransporter Proteins, Type IIa; Sodium-Phosphate Cotransporter Proteins, Type IIb
PubMed: 17595520
DOI: 10.1159/000104158