Prerequisites for combinatorial biosynthesis: evolution of hybrid NRPS/PKS gene clusters.
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Binding Sites↗
Biomedical subjects
D J Edwards
Publications and source records attributed to D J Edwards.
Effects of sustained post-traumatic shock and initial fluid resuscitation on extravascular lung water content and pulmonary vascular pressures in a porcine model of shock.
BACKGROUND: The temporal evolution of lung injury following post-traumatic shock is poorly understood. In the present study we have tested the hypothesis that manifestations of pulmonary vascular dysfunction may be demonstrable within the first hour after the onset of shock. METHODS: Twenty-nine anaesthetized pigs (mean weight 27.4 kg; (SD) 3.2) were randomly allocated to three groups: control (C, n=9), shock resuscitated with either NaCl 0.9% (S, n=10), or 4% gelatine (G, n=10). Shock was maintained for 1 h followed by fluid resuscitation with either normal saline or 4% gelatine solution. Cardiac output (CO), mean arterial pressure (MAP), mixed venous saturation (Sv(O(2))), blood lactate concentration, mean pulmonary artery pressure (MPAP), MPAP/MAP, pulmonary vascular resistance (PVR), extravascular lung water index (EVLWi), Pa(O(2))/FI(O(2)), venous admixture (Q(.)(S)/Q(.)(T)), and dynamic lung compliance (C(dyn)) were measured at baseline, beginning of shock phase, end of shock phase, and post-resuscitation. RESULTS: At the end of volume resuscitation CO was restored to control values in both shock groups. MAP remained significantly below control values (95% CI: C=70-95, S=28-52, G=45-69 mm Hg) in both shock groups. MPAP/MAP was significantly greater in both shock groups at the end of the shock phase (95% CI; C=0.15-0.24, S=0.28-0.38, G=0.32-0.42) and at the post-resuscitation phase (95% CI: C=0.12-0.30, S=0.43-0.61, G=0.32-0.49) indicating the presence of relative pulmonary hypertension. This was associated with a significant increase in PVR in Group S (F=3.9; P<0.05). There were no significant changes in Pa(O(2))/FI(O(2)), Q(.)(S)/Q(.)(T), EVLWi, or C(dyn). In a small cohort of animals a measurable increase in EVLWi (>30%) and reduction in C(dyn) (>10%) were observed. CONCLUSIONS: Pulmonary vascular injury manifesting as relative pulmonary hypertension and increased PVR may occur within the first hour after the onset of shock. These changes may not be accompanied by overt changes in oxygenation, compliance, or EVLWi. Br J Anaesth 2003; 91: 224-32
Animals↗
Electromagnetic characterisation of MR RF coils using the transmission-line modelling method.
The Transmission-Line Modelling (TLM) method is applied to the electromagnetic characterisation of RF coils and samples for magnetic resonance imaging MRI. Theoretical verification was performed using a simple surface coil. Experimental verification was performed using Alderman-Grant and birdcage coils constructed for use on a 7 T micro-imaging system. The modelling method enabled electromagnetic characteristics of frequency response, electromagnetic field generation, energy stored and power loss to be determined. From these parameters, coil resonant modes, B1 field profiles, voltages, currents, quality factor (Q), pi/2 pulse length, and the equivalent lumped-element circuit components of resistance, inductance and capacitance were calculated. Equations are presented that enable a comprehensive electromagnetic characterisation of the RF coil and sample to be achieved based on the results of the TLM simulations. The use of the TLM method is extended to include the design of safe arbitrary multi-nuclear pulse sequences such that the specific absorption rate (SAR) of tissue, and RF coil component safety limits are not exceeded.
Algorithms↗
Pharmacodynamics and pharmacokinetics of single doses of subcutaneous pegylated human G-CSF mutant (Ro 25-8315) in healthy volunteers: comparison with single and multiple daily doses of filgrastim.
Ro 25-8315 is produced by conjugation of rhG-CSF mutant with polyethylene glycol (PEG). The purpose of this study was to examine the pharmacodynamics and pharmacokinetics of Ro 25-8315 in comparison with Filgrastim (rhG-CSF). Subjects received single subcutaneous doses of Ro 25-8315 ranging from 10 to 150 microg/kg using a double-blind, randomized, placebo-controlled design. Filgrastim was administered as a single dose (5 or 10 microg/kg) and, following a 14-day washout period, daily for 7 days. Ro 25-8315 increased absolute neutrophil count (ANC) by 6- to 8-fold and CD34+ cell count more than 30-fold at the highest doses tested. Single doses (60-150 microg/kg) of Ro 25-8315 and multiple doses of Filgrastim had similar effects on ANC and CD34+, although Ro 25-8315 had a greater effect on CFU-GM. The pharmacokinetics of Ro 25-8315 were dose-dependent, with peak concentrations and area under the serum concentration-time curve (AUC) increasing 100-fold over the range of doses studied. Time to reach peak concentration (T(max)) and half-life of Ro 25-8315 averaged 20-30 hr at all doses, approximately three times longer than with Filgrastim. Adverse events were not serious and occurred with similar frequency with both products. Pegylation of rhG-CSF mutant results in more desirable pharmacokinetic properties and a longer duration of action with effective increases in ANC and measures of peripheral blood progenitor cell mobilization for at least 1 week.
Adult↗
Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin.
BACKGROUND: Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4'-phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides. PPTases have been classified according to their carrier protein specificity. In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity. However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene. RESULTS: A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp gene from S. verticillus. The svp gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters. The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution. Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S. verticillus or other Streptomyces species. As compared to Sfp, the only 'promiscuous' PPTase known previously, Svp displays a similar catalytic efficiency (k(cat)/K(m)) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP. CONCLUSIONS: PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products.
Amino Acid Sequence↗
The biosynthetic gene cluster for the anticancer drug bleomycin from Streptomyces verticillus ATCC15003 as a model for hybrid peptide-polyketide natural product biosynthesis.
The hybrid peptide-polyketide backbone of bleomycin (BLM) is assembled by the BLM megasynthetase that consists of both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules. BlmIX/BlmVIII/BlmVII constitute a natural hybrid NRPS/PKS/NRPS system, serving as a model for both hybrid NRPS/PKS and PKS/NRPS systems. Sequence analysis and functional comparison of domains and modules of BlmIX/BlmVIII/BlmVII with those of nonhybrid NRPS and PKS systems suggest that (1) the same catalytic sites appear to be conserved in both hybrid NRPS-PKS and nonhybrid NRPS or PKS systems, with the exception of the KS domains in the hybrid NRPS/PKS systems that are unique; (2) specific interpolypeptide linkers may play a critical role in intermodular communication to facilitate transfer of the growing intermediates between the interacting NRPS and/or PKS modules; and (3) posttranslational modification of the BLM megasynthetase has been accomplished by a single PPTase with a broad substrate specificity toward the apo forms of both acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs).
Antibiotics, Antineoplastic↗
Effect of buprenorphine on CYP3A activity in rat and human liver microsomes.
Buprenorphine is a partial opioid agonist available in France as an alternative to methadone in the treatment of opiate-dependent individuals. Twenty deaths have been reported in patients who have ingested buprenorphine in combination with benzodiazepines. Since buprenorphine and many benzodiazepines are CYP3A substrates, the effect of buprenorphine on CYP3A activity was examined in order to assess the likelihood of a pharmacokinetic interaction. The formation of 6beta-hydroxytestosterone was measured in dexamethasone-induced rat liver microsomes and in human liver microsomes under control conditions and in the presence of buprenorphine. Buprenorphine was found to be a weak inhibitor of CYP3A with a 50% decrease in enzyme activity occurring at a concentration of 118 microM (IC50) in human liver microsomes. IC50 was 0.3 microM for ketoconazole in the same system. Since the IC50 for buprenorphine is roughly 2000 times higher than typical plasma concentrations, this drug is unlikely to cause clinically significant inhibition of CYP3A in patients. Excessive CNS depression due to the combination of buprenorphine and benzodiazepines is most likely due to additive or synergistic pharmacologic effect unrelated to a pharmacokinetic interaction between the drugs.
Animals↗
Long-term oscillation of corticosterone following intermittent cocaine.
We have shown that repeated administration of cocaine, as well as other drugs and nondrug stressors, can induce alternating increases and decreases in several neurotransmitter and endocrine endpoints, which we call oscillation. Oscillation studies have typically used 3-4 pretreatments with cocaine or other agents, raising the question of whether oscillation lasts beyond this point. Using plasma corticosterone as our endpoint measure, we therefore inquired whether oscillation would persist across eight administrations of cocaine over a 28-day period. We report oscillation of corticosterone levels persisting across all eight cocaine groups. Our data also indicate that the degree of oscillation increases with the intertreatment interval.
Animals↗
The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase.
BACKGROUND: The structural and catalytic similarities between modular nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) inspired us to search for a hybrid NRPS-PKS system. The antitumor drug bleomycin (BLM) is a natural hybrid peptide-polyketide metabolite, the biosynthesis of which provides an excellent opportunity to investigate intermodular communication between NRPS and PKS modules. Here, we report the cloning, sequencing, and characterization of the BLM biosynthetic gene cluster from Streptomyces verticillus ATCC15003. RESULTS: A set of 30 genes clustered with the previously characterized blmAB resistance genes were defined by sequencing a 85-kb contiguous region of DNA from S. verticillus ATCC15003. The sequenced gene cluster consists of 10 NRPS genes encoding nine NRPS modules, a PKS gene encoding one PKS module, five sugar biosynthesis genes, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The substrate specificities of individual NRPS and PKS modules were predicted based on sequence analysis, and the amino acid specificities of two NRPS modules were confirmed biochemically in vitro. The involvement of the cloned genes in BLM biosynthesis was demonstrated by bioconversion of the BLM aglycones into BLMs in Streptomyces lividans expressing a part of the gene cluster. CONCLUSION: The blm gene cluster is characterized by a hybrid NRPS-PKS system, supporting the wisdom of combining individual NRPS and PKS modules for combinatorial biosynthesis. The availability of the blm gene cluster has set the stage for engineering novel BLM analogs by genetic manipulation of genes governing BLM biosynthesis and for investigating the molecular basis for intermodular communication between NRPS and PKS in the biosynthesis of hybrid peptide-polyketide metabolites.
Amino Acid Sequence↗
Analysis of messages expressed by Echinostoma paraensei miracidia and sporocysts, obtained by random EST sequencing.
A lambdaZAP Express cDNA library was constructed with mRNA obtained from immature miracidia within eggs, hatched miracidia, and sporocysts of Echinostoma paraensei. This cDNA library was amplified and 213 expressed sequence tag (EST) sequences (averaging 466 nucleotides in length) were obtained. The mean percentage of unresolved bases within the EST sequences was 0.4%, ranging from 0 to 4.6%. The 213 ESTs represent 151 unique messages. BLAST (version 2.0.8) analysis disclosed that 64 unique E. paraensei messages (42.4%) had significant similarities (BLAST score < or =e-5), at deduced amino acid or nucleotide levels, with known sequences in the nonredundant GenBank databases or the dbEST database (NCBI). The remainder, 57.6% of the unique EST-encoded messages, scored nonsignificant hits. Most of the E. paraensei messages that could be assigned a cellular role based on sequence similarities were involved in gene/protein expression. Several ESTs scored highest similarities with sequences obtained from trematode species. A total of 22,560 nucleotides present in open reading frames from ESTs that aligned with known sequences was used to determine codon usage for E. paraensei. Analysis of a subset of eight ESTs that contained full-length open reading frames did not reveal a bias in codon usage. Also, EST sequences were found to contain 3' untranslated regions with an average length of 69.9 +/- 88.4 nucleotides (n = 46). The EST sequences were submitted to GenBank/dbEST, adding to the 51 available Echinostoma-derived sequences, to provide reference information for both phylogenetic analysis and study of general trematode biology.
Animals↗
6',7'-Dihydroxybergamottin in grapefruit juice and Seville orange juice: effects on cyclosporine disposition, enterocyte CYP3A4, and P-glycoprotein.
BACKGROUND: 6',7'-Dihydroxybergamottin is a furanocoumarin that inhibits CYP3A4 and is found in grapefruit juice and Seville orange juice. Grapefruit juice increases the oral bioavailability of many CYP3A4 substrates, including cyclosporine (INN, ciclosporin), but intestinal P-glycoprotein may be a more important determinant of cyclosporine availability. OBJECTIVES: To evaluate the contribution of 6',7'-dihydroxybergamottin to the effects of grapefruit juice on cyclosporine disposition and to assess the role of CYP3A4 versus P-glycoprotein in this interaction. METHODS: The disposition of oral cyclosporine was compared in healthy subjects after ingestion of water, grapefruit juice, and Seville orange juice. Enterocyte concentrations of CYP3A4 were measured in 2 individuals before and after treatment with Seville orange juice. The effect of 6',7'-dihydroxybergamottin on P-glycoprotein was assessed in vitro. RESULTS: Area under the whole blood concentration-time curve and peak concentration of cyclosporine were increased by 55% and 35%, respectively, with grapefruit juice (P < .05). Seville orange juice had no influence on cyclosporine disposition but reduced enterocyte concentrations of CYP3A4 by an average of 40%. 6',7'-Dihydroxybergamottin did not inhibit P-glycoprotein at concentrations up to 50 micromol/L. CONCLUSIONS: 6',7'-Dihydroxybergamottin is not responsible for the effects of grapefruit juice on cyclosporine. Because the interaction did not occur with Seville orange juice despite reduced enterocyte concentrations of CYP3A4, inhibition of P-glycoprotein activity by other compounds in grapefruit juice may be responsible. Reduced enterocyte CYP3A4 by 6',7'-dihydroxybergamottin could be important for other drugs whose bioavailability is less dependent on P-glycoprotein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Cocaine-induced oscillation is conditionable.
We have recently shown that under some circumstances, sensitization produced by a stimulant such as cocaine (COC) can give way, with successive drug administrations, to alternating attenuations and reinstatements of the effect, an outcome that we have termed oscillation. Because sensitization to COC can be conditioned, we inquired whether COC-induced oscillation also was conditionable. The end point used was shock-induced hypoalgesia (paw withdrawal from a hot plate), as we have previously shown that oscillation follows initial sensitization of this measure with one to five pretreatments of 12 mg/kg (IP) of COC spaced at 1-week intervals, with the last COC injection occurring 30 min prior to the footshock. Experiment 1 indicated that a conditioned stimulus (CS)--a distinctive environment--which repeatedly had been paired with COC, would substitute for the last COC injection in sustaining the oscillatory effect. Experiment 2 showed that a previously established CS successfully substituted for all COC injections in first inducing sensitization that was then followed by oscillation. These findings strongly suggest that COC-induced oscillation shares with COC-induced sensitization, the property that both can be conditioned.
Anesthetics, Local↗
High-dose omeprazole: use of a multiple-dose study design to assess bioequivalence and accuracy of CYP2C19 phenotyping.
The objectives of this multiple-dose study were to compare the performance of a new formulation of omeprazole (40 mg) with that of an established formulation and to assess the accuracy of CYP2C19 phenotyping during high-dose chronic administration. Twenty-eight healthy subjects were randomized (1:1) to receive 40 mg of either Gasec-40 Gastrocaps (Mepha) or Antra 40 (Astra) daily for 5 days. The pharmacokinetics of omeprazole and the omeprazole/5'-hydroxyomeprazole ratio 3 hours postdose were assessed on day 5. Subjects switched formulations starting on day 6, and all measurements were repeated on day 8. Subjects with metabolic ratios greater than 6 were genotyped for CYP2C19. Gasec-40 was found to be bioequivalent to Antra based on the 90% confidence interval for AUC (102.4-111.7) and Cmax (100.6-120.7). Formulation had no effect on the ratio of omeprazole to 5'-hydroxyomeprazole, which was higher than previously reported with single 20 mg doses of omeprazole. The mean ratio did not differ between day 5 and day 8 but was highly variable: 7 of 28 subjects had more than a 2-fold difference between assessments. In four individuals identified by genotype as extensive metabolizers (EMs), phenotype could not be clearly assigned. The relative bioavailability of omeprazole can be accurately assessed using this multiple-dose study design. Chronic administration of 40 mg doses of omeprazole shifts the metabolic ratio in EMs toward that in poor metabolizers (PMs), apparently because of the nonlinear metabolic clearance of the drug. The assignment of phenotype in patients receiving chronic high-dose omeprazole treatment should be interpreted with caution.
2-Pyridinylmethylsulfinylbenzimidazoles↗
Forging partnerships in care.
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Community Networks↗
Impact of third molar removal on demands for postoperative care and job disruption: does anaesthetic choice make a difference?
A prospective cohort study was undertaken to investigate the influences of anaesthetic modality and surgical difficulty on social reintegration and demands on health services after third molar removal. The study was undertaken at the Oral and Maxillofacial Surgery Department, Cardiff Dental Hospital. Of 444 patients, 266 (60%) had their third molars removed. The main outcome measures included anaesthetic modality, surgical difficulty (WHARFE scores), utilisation of health services, effects on work, school and home life. In all, 101 (40%) patients were treated under local anaesthesia (LA) +/- intravenous (i.v.) sedation and 165 (60%) under general anaesthesia (GA); 81 (49%) as inpatients and 84 (51%) as day cases. Of these patients, 38 (14%) returned to the hospital and 74 (28%) utilised primary care services postoperatively in addition to a standard review appointment. Patients treated under GA made more demands on primary care services (chi 2 = 6.41, df = 2, P < 0.05) and took more time away from work (P < 0.05). Patients underestimated the time they needed to recover. There was similar disruption to job, college and home life. There were no links between disruption and particular anaesthetic modalities and surgical difficulty. Surgery under GA was linked to increased postoperative demands on primary care, but not secondary care, and to longer job disruption. This could not fully be attributed to surgical difficulty.
Adolescent↗
Give logistics its own place in the price equation.
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Contract Services↗
Interactions of phenol and m-cresol in the insulin hexamer, and their effect on the association properties of B28 pro --> Asp insulin analogues.
Insulin's natural tendency to form dimers and hexamers is significantly reduced in a mutant insulin B28 Pro --> Asp, which has been designed as a monomeric, rapid-acting hormone for therapeutic purposes. This molecule can be induced to form zinc hexamers in the presence of small phenolic derivatives which are routinely used as antimicrobial agents in insulin preparations. Two structures of B28 Asp insulin have been determined from crystals grown in the presence of phenol and m-cresol. In these crystals, insulin exists as R6 zinc hexamers containing a number of phenol or m-cresol molecules associated with aromatic side chains at the dimer-dimer interfaces. At the monomer-monomer interfaces, the B28 Pro --> Asp mutation leads to increased conformational flexibility in the B chain C termini, resulting in the loss of important intermolecular van der Waals contacts, thus explaining the monomeric character of B28 Asp insulin. The structure of a cross-linked derivative of B28 Asp insulin, containing an Ala-Lys dipeptide linker between residues B30 Ala and A1 Gly, has also determined. This forms an R6 zinc hexamer containing several m-cresol molecules. Of particular interest in this structure are two m-cresol molecules whose binding disrupted the beta-strand in one of the dimers. This observation suggests that the cross-link introduces mechanical strain on the B chain C terminus, thereby weakening the monomer-monomer interactions.
Aspartic Acid↗
Oscillatory effects of repeated morphine on shock-induced hypoalgesia and beta-endorphin.
Recent research indicates that the sensitization that results from repeated drug or non-drug stress exposure may develop into a pattern of alternating increases and decreases (i.e., oscillation) in response to each subsequent stressor exposure. Oscillation, with or without prior sensitization, has been observed for a number of drug and non-drug stressors, and for various neurochemical and endocrine endpoints. The present studies investigated whether oscillation also occurs in the behavioral and endocrine effects of repeated morphine treatment and if a drug that normalizes the mood swings of bipolar disorder in humans will also attenuate drug oscillation in this animal model. In the first experiment, rats were given 1-5 pretreatments with morphine (15 mg/kg, i.p.), separated by 1-week intervals with the last injection occurring 1 hour prior to being tested for stressor-induced (i.e., 5 seconds, 2-mA electric footshock) hypoalgesia, as measured by latency to paw-lift or jump from a hot-plate. Plasma beta-endorphin also was measured. The second experiment replicated the behavioral findings of the first study and, in addition, assessed the effect of continuous lithium chloride, in the drinking water, on morphine-induced oscillation. Caffeine was used as a partial control for the lithium. The results were that one injection of morphine enhanced stress-induced hypoalgesia and subsequent morphine administrations resulted in oscillation. Beta-endorphin exhibited sensitization but not oscillation, suggesting that it did not mediate oscillation of the behavioral response. In addition, lithium, but not caffeine, eliminated oscillations of the behavioral response without affecting its initial enhancement.
Analysis of Variance↗