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J E Murphy

Publications and source records attributed to J E Murphy.

At least 55 records · Page 3Linked to original sources
PubMed1994

Clathrin assembly protein AP-3 is phosphorylated and glycosylated on the 50-kDa structural domain.

AP-3 (AP180) in rat sympathetic neurons maintained in culture was analyzed by pulse-chase labeling with [35S]methionine to look for post-translational modifications. At early times, two lower molecular weight precursors of the mature species were detected. By 10 min, all of the AP-3 was found in the mature form which is stable for at least 9 h. We show here that at least one of these processing events is due to the addition of O-linked N-acetylglucosamine (GlcNAc) which is present on the mature form of the protein. Wheat germ agglutinin, a GlcNAc-specific probe, bound to AP-3 and the binding was blocked by excess GlcNAc but not by excess mannose. Purified AP-3, and AP-3 in coated vesicles derived from bovine brain, served as substrates for beta-D-galactosyltransferase which is specific for terminal GlcNAc residues. Analysis of the disaccharide released by beta-elimination indicated that single GlcNAc residues are attached to AP-3 through an O-glycosidic linkage to threonine or serine residues. In vivo 32P-labeled AP-3, the result of serine phosphorylation (Keen, J. H., and Black, M.M. (1986) J. Cell Biol. 102, 1325-1333), bound to wheat germ agglutinin-Sepharose indicating that phosphorylation and glycosylation can occur simultaneously on the same molecule. Both modifications have been mapped to the central 50-kDa structural domain that is responsible for the anomalous migration of AP-3. Consistent with localization to the nonclathrin binding domain, the O-GlcNAc modification does not play a discernible role in the interaction of AP-3 with clathrin.

Acetylglucosamine↗
PubMed1994

Forced integration of Moloney murine leukemia virus DNA with a mutant integration site occurs through recombination with VL30 DNA.

The integration of retroviral DNA to form the provirus requires the presence of short inverted repeat sequences at the termini of the linear viral DNA. We have previously described the construction of a mutant of Moloney murine leukemia virus, carrying an 8-bp deletion in the U5 terminus, that is defective in establishing the integrated provirus. We have now used a selectable marker to allow recovery of rare proviruses formed after transduction from NIH/3T3 cells by retroviral vectors carrying this mutation. Analysis of two such proviruses showed that both were recombinants between the vector and VL30 DNA and arose such that the VL30 sequences could provide an intact terminal sequence for integration.

3T3 Cells↗
PubMed1994

Why are mammalian alkaline phosphatases much more active than bacterial alkaline phosphatases?

Mammalian alkaline phosphatases are 20-30-fold more active than the corresponding bacterial enzymes even though their amino acid sequences are 25-30% absolutely conserved. In the active-site region there are two noticeable differences between the sequences of the bacterial and mammalian enzymes. In the Escherichia coli enzyme positions 153 and 328 are Asp and Lys, respectively, but in the mammalian enzymes His is observed at both of these positions. Site-specific mutagenesis, genetic and X-ray crystallographic data, which will be summarized here, suggest that the His substitutions at positions 153 and 328 are primarily responsible for the differences in properties between the bacterial and mammalian alkaline phosphatases.

Alkaline Phosphatase↗
PubMed1994

Delayed effect of wheat starch in foods on the intraoral demineralization of enamel.

There is considerable evidence for an association between dental caries and food starches. However, the intraoral utilization of starch may be quite complex, giving rise to conflicting results. Demineralization induced by unsweetened cookies was examined in an intraoral model system that utilized palatal appliances containing blocks of bovine enamel. The enamel surfaces were covered with either a filter paper disc to trap sugars or a layer of Streptococcus mutans to metabolize the sugars and bring about enamel demineralization. Demineralization was determined as an increase in porosity with respect to iodide ions (delta Ip). Measurements revealed a rapid elevation and maintenance of high levels of maltose in the plaque space after ingestion of the unsweetened or sweet cookies. Entrapped food particles appeared to serve as a reservoir of maltose. Unsweetened cookies brought about enamel demineralization, but the pH of the streptococcal plaque fell slowly, and the initiation of demineralization was delayed. Thus, delta Ip and plaque pH were -0.3 +/- 1.3 U and 6.1 +/- 0.3, respectively, after 15 min. The delay was shown to be related to the need to induce the acidogenic streptococci to metabolize maltose. Once induced, delta Ip rose rapidly and reached a maximum at 45 min. Sweet cookies released sucrose and maltose and brought about a rapid onset of demineralization. In summary, the data demonstrated (1) that maltose was released rapidly from unsweetened cookie particles and diffused into the plaque space of the model system and (2) that maltose-dependent demineralization of enamel required time for the induction of the streptococcal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Conversion of a magnesium binding site into a zinc binding site by a single amino acid substitution in Escherichia coli alkaline phosphatase.

The replacement of aspartic acid by histidine at position 153 in Escherichia coli alkaline phosphatase results in a mutant enzyme that is remarkably similar to certain mammalian alkaline phosphatases that are activated by magnesium in a time-dependent fashion. These mammalian alkaline phosphatases have histidine at the position corresponding to 153 of the E. coli sequence. Here we report the three-dimensional structure of the mutant E. coli alkaline phosphatase with histidine at position 153. The structure reveals that the octahedral magnesium binding site has been converted to a tetrahedral zinc binding site with an imidazole ring nitrogen of His-153 as one of the ligands to the zinc. The alteration in metal binding caused by the mutation could explain the origin of the magnesium activation observed with the mammalian alkaline phosphatases. The structure also reveals differences in the mode of phosphate binding, explaining the enhanced phosphate affinity and the reduced activity of the mutant enzyme in the presence of zinc.

Alkaline Phosphatase↗
PubMed1993

Magnesium in the active site of Escherichia coli alkaline phosphatase is important for both structural stabilization and catalysis.

Site-specific mutagenesis was used to explore the roles of the side chains of residues Lys-328 and Asp-153 in Escherichia coli alkaline phosphatase. The D153H enzyme exhibits a 3.5-fold decrease in activity at pH 8.0 compared to that of the wild-type enzyme, while a double mutant D153H/K328H exhibits a 16-fold decrease in activity under these conditions. However, the Km values for both enzymes, employing the substrate p-nitrophenyl phosphate, are lower than the value for the wild-type enzyme. The Ki for phosphate, which is pH- and Mg(2+)-dependent, is decreased for the D153H enzyme and increased for the D153H/K328H enzyme. Relative to the wild-type enzyme, both mutant enzymes bind Mg2+ more weakly and undergo a time-dependent activation induced by Mg2+. The half-time of the activation process is independent of the Mg2+ concentration, indicating that the activation most probably involves a conformational change. The pH versus activity profiles of both enzymes are altered relative to that of the wild-type enzyme and exhibit greatly enhanced activity, relative to that of the wild-type enzyme, at high pH values. The pre-steady-state kinetics for the D153H and D153H/K328H enzymes exhibit a transient burst of product formation at pH 8.0, under conditions at which the wild-type enzyme exhibits no transient burst, indicating that at pH 8.0 the hydrolysis of the covalent enzyme-phosphate complex is rate-determining and not the release of phosphate from the noncovalent enzyme-phosphate complex as is observed for the wild-type enzyme. Therefore, these mutations are directly influencing catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1993

Kinetic analysis of furan biotransformation by F-344 rats in vivo and in vitro.

Furan is both hepatotoxic and hepatocarcinogenic in rats. The kinetics of furan biotransformation by male F-344 rats were studied in vivo and in vitro in order to understand target tissue dosimetry. A physiologically based pharmacokinetic (PBPK) model for furan in rats was developed from gas uptake studies using initial furan concentrations of 100, 500, 1050, and 3850 ppm. Tissue partition coefficients for furan were determined in vitro using vial equilibration techniques. Furan gas uptake kinetics in vivo were described by a single saturable process with a Vmax of 27.0 mumol/hr/250 g rat and a KM of 2.0 microM. Furan metabolism in vivo was inhibited by pyrazole. The furan PBPK model adequately simulated blood and liver furan concentrations following 4-hr inhalation exposures to 52, 107, and 208 ppm furan. The biotransformation of furan was studied in freshly isolated rat hepatocytes in vitro and compared to biotransformation in vivo. Furan biotransformation by isolated rat hepatocytes exhibited a KM of 0.4 microM and a Vmax of 0.018 mumol/hr/10(6) cells. Inhibition and induction studies indicated that cytochrome P450 was the catalyst of furan oxidation. Acetone pretreatment of the rats produced a five-fold increase in the rate of the hepatocyte oxidation of furan, suggesting an important role for cytochrome P450 2E1. The Vmax determined in hepatocytes in vitro extrapolated to 23.0 mumol/hr/250 g rat, assuming 128 x 10(6) hepatocytes/g liver. Incorporation of the in vitro hepatocyte kinetic parameters into the PBPK model for furan accurately simulated in vivo pharmacokinetics. These results suggest that freshly isolated hepatocytes are a valuable in vitro system for predicting chemical pharmacokinetics in vivo.

Animals↗
PubMed1993

Mutational analysis of the sequences at the termini of the Moloney murine leukemia virus DNA required for integration.

The insertion of retroviral DNA into the genome of the infected cell to form the integrated provirus requires the presence of short inverted repeat (IR) sequences at the viral DNA termini. To examine the sequence requirements at the IRs for integration of the Moloney murine leukemia virus, we generated a series of mutants with deletion and substitution mutations and tested their ability to replicate and form proviruses in vivo. The experiments did not detect evidence of a requirement for sequences outside the IRs, and only a very loose requirement for the IR sequences, with many point mutations well tolerated. Examination of the mutants suggests that bases very near the terminus and those approximately one turn of the DNA helix away from the terminus are important for recognition by the integrase function.

3T3 Cells↗
PubMed1993

Vancomycin pharmacokinetics in neonates and infants: a retrospective evaluation.

OBJECTIVE: To evaluate the frequency with which current loading and maintenance vancomycin dosages achieve target serum concentrations based on pharmacokinetic parameters obtained after the initial dose. Also, to identify the daily vancomycin dosage necessary to achieve target serum concentrations at steady-state and to determine if any relationships exist between vancomycin pharmacokinetic parameters and various patient characteristics. SETTING: Neonatal intensive care unit (NICU) at Georgia Baptist Medical Center. PATIENTS/METHODS: Twenty-three infants with suspected or documented gram-positive infection who received intravenous vancomycin between July 1990 and November 1991 were included in this retrospective analysis. Gestational age range from 23 to 41 weeks and postconceptional age (PCA) at the time of the study ranged from 26 to 46 weeks. Vancomycin therapy was initiated with a loading dose of 15 mg/kg, followed by a maintenance dosage of 20-30 mg/kg/d, which was usually given as 10 mg/kg q8-12h. All vancomycin doses were administered using a syringe pump. Peak and trough serum concentrations were obtained following the first dose. Vancomycin pharmacokinetic parameters were determined using a one-compartment model. Infants receiving indomethacin within two weeks prior to study were analyzed separately (group 2, n = 4). All other infants were included in group 1 (n = 19). RESULTS: For group 1, vancomycin clearance (Cl), volume of distribution (Vd), and half-life were (mean +/- 1 SD) 0.072 +/- 0.032 L/kg/h, 0.52 +/- 0.08 L/kg, and 5.6 +/- 1.6 hours, respectively. For both groups, loading doses provided 1-hour postinfusion peak concentrations of 25-35 mg/L in one of every two infants studied, whereas only three percent of initial maintenance doses were projected to provide desired peak and trough concentrations at steady-state. For group 1, the mean daily dosage necessary to provide target peak (25-35 mg/L) and trough (5-10 mg/L) concentrations at steady-state was larger than that initially prescribed (29.6 +/- 13.1 vs. 22.2 +/- 4.7 mg/kg/d). For group 2, the mean daily dosage required to achieve target peak and trough concentrations at steady-state was smaller than that initially prescribed (14.8 +/- 4.3 vs. 20.0 +/- 0.1 mg/kg/d) and was exactly half of that required for group 1. Excellent correlations were observed between PCA and vancomycin Cl (L/h) (r = 0.92; p < 0.0001), body weight and Vd(L) (r = 0.94; p < 0.0001), body weight and vancomycin Cl (L/h) (r = 0.85; p < 0.0001), PCA and Vd (L) (r = 0.89; p < 0.0001), and body surface area and Vd (L) (r = 0.93; p < 0.0001) for group 1. Moderate correlations were also noted between PCA and Cl relative to body weight (L/kg/h), postnatal age and Cl (L/kg/h), and PCA and vancomycin dosage requirements (mg/kg/d). No linear correlation was observed between any patient characteristic and Vd standardized for body weight. CONCLUSIONS: Our data demonstrate the need for a more accurate method of estimating initial vancomycin dosage requirements in this NICU population. Although some of the relationships revealed in this study could be used to determine vancomycin dosage for infants in the range of approximately 30-36 weeks PCA, we hesitate to suggest this approach presently because of the potential limitations of our study design. Further prospective study is needed to confirm these observations. In addition, further study is necessary to describe the time course of the interaction between vancomycin and indomethacin in infants with successful and unsuccessful closure of their patent ductus arteriosus.

Female↗
PubMed1992

Inositol hexakisphosphate receptor identified as the clathrin assembly protein AP-2.

To clarify the function of the receptor binding protein for inositol hexakisphosphate (IP6), we obtained a partial amino acid sequence from the purified protein and a partial nucleotide sequence from a cDNA clone of the gene. The sequences are essentially identical to those of the alpha-subunit of the clathrin assembly protein AP-2. The IP6 receptor protein analyzed by SDS-PAGE contains a series of subunits which are the same as those of AP-2. Antibodies to AP-2 react with the IP6 receptor protein in immunoblot analysis.

Adaptor Proteins, Vesicular Transport↗
PubMed1992

Recognition sites for clathrin-associated proteins AP-2 and AP-3 on clathrin triskelia.

AP-2 and AP-3 are cellular proteins that drive the in vitro polymerization of clathrin triskelia into cage structures. The interaction of these two types of assembly proteins (APs) with preassembled clathrin cages has been studied in order to identify the sites on the triskelia required for binding. Comparing binding of the APs to intact or to proteolytically clipped cages, we attempted to distinguish between binding to the terminal domain, the globular end of the heavy chain, and binding to the hub of the clathrin triskelia, the portion that remains assembled after trypsin treatment. AP-3 binds to intact clathrin cages but not to those that were treated with trypsin. AP-3 also bound to cages consisting solely of clathrin heavy chains; proteolysis of these cages also eliminated AP-3 binding. In addition, AP-3 did not bind to either isolated hubs or terminal domains that had been immobilized on Sepharose. These data indicate that clathrin light chains are not required for binding of AP-3, and that neither terminal domain nor hubs alone will suffice. However, an intact heavy chain is both necessary and sufficient for the binding of AP-3. Previous work has demonstrated one binding site for AP-2 on proteolyzed cages containing only clathrin hubs; the existence of a second binding site associated with the terminal domain was hypothesized. Here we provide direct evidence for recognition by AP-2 of isolated terminal domains immobilized on Sepharose and show that the core of the AP-2 molecule is responsible for this interaction. These results provide the first demonstration of a functional role for the conserved terminal domain of the clathrin heavy chain.

Adaptor Proteins, Vesicular Transport↗
PubMed1992

Effect of education on the appropriateness of serum drug concentration determination.

The purpose of this study was to document the effect of education on the appropriateness of serum drug concentration (SDC) collection. This study included a period of education for hospital personnel involved in the acquisition of drug levels. These included nursing staff, lab personnel, house-staff (residents/interns), ward clerks, and pharmacists. This study included patients receiving aminoglycoside and/or vancomycin while on the general medicine service during the specified study periods. Patients receiving less than 72 h of antimicrobial therapy and patients transferred to or from the surgery service were excluded from the study. A retrospective pre- and posteducation comparison using Chi-square analysis was made to determine whether the percentage of appropriate and useful SDCs was improved through education. There was a significant difference (p less than 0.005) between the percentage of appropriate SDCs in the pre- and posteducation period. As a result of the study, a change in procedure for ordering SDCs was initiated, using an educational program, in order to continue the trend of appropriate SDCs at the Veterans Administration.

Aminoglycosides↗
PubMed1992

Use of theophylline in neonates as an aid to ventilator weaning.

Respiratory diseases are major causes of morbidity and mortality in premature neonates. Theophylline has been utilized as an adjunct in facilitating ventilator weaning and in the management of apnea with or without bradycardia. Patient characteristics associated with improved outcome from theophylline have not been determined. The purpose of this study was to evaluate parameters associated with improved outcome in neonates with respiratory diseases receiving theophylline. The study population consisted of premature neonates that were studied retrospectively. Criteria for entry into the study were (1) less than 40 weeks gestation, (2) a diagnosis of respiratory distress syndrome (RDS), apnea of prematurity, hyaline membrane disease (HMD), or bronchopulmonary dysplasia (BPD), (3) dependence on intermittent mandatory ventilation, (4) failure to wean from the ventilator 24 h or more before the study, or (5) receiving theophylline. In this study, we found no correlations between time to wean from the ventilator and postnatal age at the time theophylline was initiated, 5-min APGAR score, and final theophylline serum concentration before complete weaning from the ventilator. However, there were significant negative correlations between birthweight and gestational age with respect to time to wean from the ventilator. The average theophylline serum concentration before weaning from the ventilator for this population of neonates was approximately 5-10 micrograms/ml, indicating that theophylline is not beneficial as an aid to ventilator weaning at serum concentrations < 10 micrograms/ml.

Apgar Score↗
PubMed1992

A mutation at one end of Moloney murine leukemia virus DNA blocks cleavage of both ends by the viral integrase in vivo.

The integration of retroviral DNA proceeds through two steps: trimming of the termini to expose new 3' OH ends, and the transfer of those ends to the phosphates of target DNA. We have examined the ability of the Moloney murine leukemia virus integrase protein (IN) to trim the termini of the preintegrative DNA of mutant viruses with alterations in the U3 inverted repeat. The mutant terminus of one replication-defective viral DNA, containing a 7-bp deletion in the U3 inverted repeat, was not trimmed to produce the normal recessed end. Remarkably, the other terminus of this mutant DNA was also not trimmed, even though its sequence is wild type. This finding suggests that the IN protein requires the presence of two good ends before becoming properly activated to trim either one.

Base Sequence↗
PubMed1991

Clathrin assembly protein AP-3. The identity of the 155K protein, AP 180, and NP185 and demonstration of a clathrin binding domain.

Three independently isolated clathrin-associated proteins have been reported that have molecular weights of approximately 155,000-185,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: the 155K protein (Keen, J. H., and Black, M. M. (1986) J. Cell Biol. 102, 1325-1333), AP 180 (Ahle, S., and Ungewickell, E. (1986) EMBO J. 5, 3143-3149), and NP185 (Kohtz, D. S., and Puszkin, S. (1988) J. Biol. Chem. 263, 7418-7425). Using two-dimensional isoelectric focusing polyacrylamide gel electrophoresis and one- and two-dimensional immunoblots with two different monoclonal antibodies, we show that these three proteins are identical. The term AP-3 is used to denote this protein. A preliminary analysis of the domain structure of AP-3 was done by controlled proteolysis. Trypsin treatment of AP-3 yields two distinct classes of products. The larger fragments obtained (100,000-135,000 apparent Mr) are acidic and behave anomalously on gel electrophoresis, yielding aberrantly high Mr and exhibiting poor dye binding; these characteristics are shared with intact AP-3. Trypsin also generates a smaller neutral species of approximately 30,000 Da which migrates appropriately on sodium dodecyl sulfate-gel electrophoresis, binds dye comparatively strongly, and behaves as a monomeric globular species in solution. In addition, this species, which is also released by a variety of other proteases, binds specifically and reversibly to clathrin-Sepharose, identifying it as a clathrin recognition domain.

Adaptor Proteins, Vesicular Transport↗
PubMed1991

Effect of monitoring drug concentrations through lines used to administer the drugs: an in vitro study.

Elevated drug concentration (DC) can be caused by intentional or iatrogenic overdoses and sampling technique errors. This study examined, in vitro, technique factors that can cause and prevent false elevations of DCs when drugs are administered through the catheter lines from which blood samples are later taken for DC analysis. Digoxin, aminophylline, and phenytoin were administered through central catheters in concentrations simulating those used clinically. Drug solution remained in the lumen for a time similar to that encountered clinically, then either remained in the tubing or was flushed out with 5 ml of normal saline (NS). After 6 h, a 5-ml sample was withdrawn for DC analysis (the tip of the catheter placed in NS, which represented blood supply). Prior to final sample withdrawal, 5 ml of NS was drawn through half of the lines and discarded to simulate methods used to prevent contamination. Thus, various flushing/no flushing and sample discarding/no discarding techniques were analyzed. When diluted concentrations of drug were administered, minor artifactual DCs were observed unless the line was not flushed or sample fluid discarded prior to final sampling. With undiluted drug administration, only flushing after the dose and discarding a sample prior to final sampling prevented artifactual DCs. Clinicians should be aware of the potential for such artifactual elevations of DCs and should encourage techniques that prevent their occurrence.

Aminophylline↗
PubMed1991

A statewide survey of pharmacokinetic service provision in Georgia.

A survey of pharmacokinetic service (PKS) provision and characteristics of the service was conducted in Georgia's 223 hospitals. The survey's questionnaire, returned by 133 (59.6%) institutions, showed that 23.3% currently had a PKS and that 47% of those without a PKS had plans to establish one in the future. Services were primarily provided by pharmacists (93%) who were certified to provide the consultations in only 40% of the institutions and the PKS was run through either the pharmacy (93%) or pathology laboratory. All services used calculators and/or computers with a variety of software programs to assist in pharmacokinetic evaluations. Patients were charged for the service in 38% of the institutions.

Adolescent↗