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Biomedical subjects

R S Phillips

Publications and source records attributed to R S Phillips.

At least 181 records · Page 10Linked to original sources
PubMed1990

Mechanism of binding of substrate analogues to tryptophan indole-lyase: studies using rapid-scanning and single-wavelength stopped-flow spectrophotometry.

We have examined the binding of oxindolyl-L-alanine, (3R)-2,3-dihydro-L-tryptophan, L-homophenylalanine, and N1-methyl-L-tryptophan to tryptophan indole-lyase (tryptophanase) from Escherichia coli by using rapid-scanning and single-wavelength stopped-flow kinetic techniques. Rate constants for the reactions were determined by fitting the concentration dependencies of relaxations to either linear (pseudo-first-order) or hyperbolic (rapid second-order followed by slow first-order) equations. The reaction with oxindolyl-L-alanine forms a quinonoid intermediate that exhibits a strong peak at 506 nm. This species is formed more rapidly than with the other analogues (84.5 s-1) and is reprotonated very slowly (0.2 s-1). Reaction with L-homophenylalanine also forms a quinonoid intermediate with a strong peak at 508 nm, but the rate constant for its formation is slower (6.9 s-1). The reaction with L-homophenylalanine exhibits a transient intermediate absorbing at about 340 nm that decays at the same rate as the quinonoid peak forms and that may be a gem-diamine. Tryptophan indole-lyase reacts with (3R)-2,3-dihydro-L-tryptophan much more slowly to form a moderately intense quinonoid peak at 510 nm, and a transient intermediate absorbing at about 350 nm is also formed. The species formed in the reaction of N1-methyl-L-tryptophan exhibits a peak at 425 nm and a very weak quinonoid absorption peak at 506 nm, which is formed at less than 4 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1990

The environments of Trp-248 and Trp-330 in tryptophan indole-lyase from Escherichia coli.

The two tryptophan residues, Trp-248 and Trp-330, in tryptophan indole-lyase (tryptophanase) from E. coli have been separately mutated to phenylalanine using site-directed mutagenesis. Both single tryptophan mutant enzymes have full catalytic activity, but exhibit different fluorescence and near-UV circular dichroism spectra. These results indicate that Trp-330 is more deeply buried than is Trp-248, and is in a more asymmetric environment. Neither residue reacts with N-bromosuccinimide (NBS), although tryptophan indole-lyase is inactivated by NBS. These results demonstrate that the tryptophan residues in tryptophan indole-lyase are not catalytically essential.

Binding Sites↗
PubMed1990

Oxygenation of fluorinated tyrosines by mushroom tyrosinase releases fluoride ion.

The reactions of 2-fluoro- and 3-fluoro-L-tyrosine with mushroom tyrosinase have been investigated. Both fluorinated tyrosines are good substrates for tyrosinase, with Vmax and Vmax/Km values similar to those for L-tyrosine. Oxygenation of 2-fluorotyrosine is regioselective, and only 6-fluorodopa was detected by HPLC in reaction mixtures. Oxygenation of both isomers of monofluorotyrosine results in fluoride ion production in the absence of ascorbic acid; however, 2-fluorotyrosine also produces fluoride in the presence of ascorbic acid. These results are consistent with previous studies demonstrating rapid intramolecular cyclization of nascent 6-fluorodopaquinone (M.E. Rice, B. Moghaddan, C.R. Creveling, and K.L. Kirk, 1987, Anal. Chem. 59, 1534-1538), which is competitive with reduction by ascorbate, resulting in elimination of the aromatic fluorine as fluoride ion.

Basidiomycota↗
PubMed1990

Early appearance of variant parasites in Plasmodium chabaudi infections.

Previous studies have shown that the recrudescence parasitaemias seen in mice infected with Plasmodium chabaudi AS strain are antigenically different from the infecting parent population. Antigenic differences between recrudescent and parent populations were demonstrated in a passive transfer assay. In the present study, using the same assay system, it has been shown that in some mice, variant parasites (i.e. different from the parent population) can be detected at a time when the primary parasitaemia is still patent but in remission. This is the first report in Plasmodium of variant parasites being detected during the course of a patent primary parasitaemic episode of an infection initiated with a cloned line.

Animals↗
PubMed1990

Duodenal obstruction: a complication of percutaneous endoscopic gastrostomy tube migration.

Percutaneous endoscopic gastrostomy (PEG) is now a well-established technique for providing long-term enteral feeding. The procedure is being increasingly utilized and, although generally well tolerated, it may be associated with complications. We report two patients in whom a replacement Foley balloon tip catheter migrated into the small bowel and caused obstruction. The diagnosis, management, and preventive measures of tube migration are discussed.

Aged↗
PubMed1990

Oral dissolution therapy for cholelithiasis: mix and match.

The authors conducted a prospective, randomized trial of chenodeoxycholic and ursodeoxycholic acid versus ursodeoxycholic acid alone in patients with cholelithiasis to determine their efficacy for dissolution of gallstones. One hundred and twenty patients with radiolucent gallstones, less than or equal to 15 mm and who had a functioning gallbladder were enrolled. The patients were divided into two groups based on the diameter of their largest stones. Seventy patients had stones larger than 5 mm but less than 15 mm, whereas 50 patients had stones that measured 5 mm or less. The patients were randomly assigned to treatment with chenodeoxycholic acid plus ursodeoxycholic acid (5 mm/kg of each) or ursodeoxycholic acid (10 mm/kg) alone. Oral cholecystography, plain abdominal x-rays, and ultrasonography of the gallbladder were done at 6, 12, and 24 months. Dissolution was deemed to be complete if not stones were visualized on two examinations. partial dissolution was defined as a 50% reduction in stone size and/or number. Stones that were not detected by cholecystography but still detected during ultrasonography were considered to be partially dissolved. Plasma triglycerides, serum cholesterol, HDL, and serologic liver function tests were determined at 1, 3, 6, 12, 18, and 24 months. In a select group of patients, bile-rich duodenal aspirates were aspirated and analyzed for biliary lipid contents. In the group with small stones, defined as less than or equal to 5 mm, complete stone dissolution occurred significantly more often utilizing combination therapy at 6 months (52% vs 24%), and this trend persisted, although no longer significant, at 12 and 24 months. Combination therapy also achieved an improved rate of dissolution for large stones within 6 months; however, this did not persist at 12 and 24 months. Although not statistically significant, stone calcification occurred less often with combined therapy. All treatment regimens were well tolerated, with only minor changes in bowel habits and mild elevations in serum transaminase levels. Serum lipid levels did not change with either therapy. The authors concluded that the combination of chenodeoxycholic acid and ursodeoxycholic acid was the preferred therapy for gallstone dissolution, because it dissolves stones more rapidly, with a lower incidence of stone calcifications, and thus might reduce the long-term cost of treatment.

Chenodeoxycholic Acid↗
PubMed1989

Evidence that cysteine 298 is in the active site of tryptophan indole-lyase.

Escherichia coli tryptophan indole-lyase (tryptophanase) mutants, with cysteine residues 294 and 298 selectively replaced by serines, have been prepared by site-directed mutagenesis. Both mutant enzymes are highly active for beta-elimination reactions measured with both L-tryptophan and S-(o-nitrophenyl)-L-cysteine. The Cys-294----Ser mutant enzyme is virtually identical to the wild type with respect to pyridoxal phosphate binding (KCO = 2 microM), cofactor absorption spectrum (lambda max = 420 and 337 nm) and pH dependence (pK alpha = 7.3), pH profile for catalysis, and rate of bromopyruvic acid inactivation. In contrast, the Cys-298----Ser mutant enzyme exhibits a reduced affinity for pyridoxal phosphate (KCO = 6 microM), a shift in the cofactor absorption spectrum to 414 nm and an altered pK alpha = 8.5, an alkaline shift in the pH profile for catalysis, and resistance to inactivation of the apoenzyme by bromopyruvic acid. The C298S mutant enzyme (wherein cysteine 298 is altered to serine) also undergoes an isomerization to an unreactive state upon storage at 4 degrees C. These results demonstrate that the sulfhydryl groups of Cys-294 and Cys-298 are catalytically nonessential. However, these data suggest that Cys-298 is located within or very near the active site of the enzyme and is the reactive cysteine residue previously observed by others.

Base Sequence↗
PubMed1989

The use of murine feeder cells in the cultivation of Plasmodium falciparum asexual blood stages.

Increased multiplication rates were observed in asexual erythrocytic stages of Plasmodium falciparum grown in the presence of a feeder-cell layer of mouse peritoneal wash cells (PWCs) using the candle-jar method of Trager and Jensen (1977). This held true for both new and established isolates of the parasite. When the PWC population was separated into adherent and non-adherent fractions, the adherent PWC population promoted an increase in parasite growth but the non-adherent population did not. An increase in parasite multiplication was not promoted by PWC-conditioned medium.

Animals↗
PubMed1989

Should women be tested for gonococcal infection of the cervix during routine gynecologic visits? An economic appraisal.

PURPOSE, PATIENTS, AND METHODS: We used decision analysis to calculate the economic implications of routine testing for Neisseria gonorrhoeae infection of the cervix. This study compared performing endocervical cultures in all women seeking routine gynecologic care to a no-test strategy. Estimates derived from the medical literature were varied in a sensitivity analysis, and a Monte Carlo technique was used to incorporate the estimates of experts into the analysis. RESULTS: We found that routinely testing for gonorrhea reduced overall medical costs when the prevalence of infection exceeded 1.5 percent. This result was most sensitive to variation in the cost of the culture and the assumption that treatment prevents adverse sequelae. Reducing the estimated cost of the culture from $9 to $5 decreased the threshold prevalence from 1.5 to 0.8 percent. When we considered the variation in estimates of experts, the threshold prevalence increased from 1.5 to 2.1 percent. CONCLUSION: We conclude that a strategy of testing and treating selected women with risk factors for gonorrhea will not only reduce the likelihood of the development of serious adverse sequelae but will also result in a reduction of medical costs.

Adult↗
PubMed1989

Chlamydia trachomatis cervical infection in women seeking routine gynecologic care: criteria for selective testing.

PURPOSE: Chlamydia trachomatis infection is one of the most common sexually transmitted diseases. Developing methods to identify patients who are at higher risk would enable clinicians to perform selective testing for C. trachomatis infection, which may reduce morbidity, sequelae, and overall costs. Risk factors for C. trachomatis infection of the cervix in women seen for routine gynecologic care were therefore analyzed. PATIENTS AND METHODS: Extensive clinical data and cervical cultures were obtained from 1,141 women between the ages of 18 and 50. Multivariate techniques were used to develop and prospectively test a model to predict infection. RESULTS: In the model development series of 530 patients seen in a hospital-based practice, 24 (4.5%) had a positive culture result. In the validation series of 611 patients seen in private practice, 17 (2.8%) had positive culture results. The majority of infected patients (61%) were asymptomatic. For sexually active women 18 to 35 years of age, the optimal predictive model was based on three factors: level of education (high school or less), a sex partner with other partners during the prior three months, and endocervical bleeding induced by the initial swab. In the validation series, the prevalence of infection for women with one or more of these factors was 5.8% (95% confidence interval, 4.0 to 8.6) compared with 0.9% in patients without these factors (p = 0.005). CONCLUSION: Clinical information can be used in a low-prevalence population to predict a patient's risk for C. trachomatis infection and to select patients for testing.

Adolescent↗
PubMed1989

Interventions to prevent readmission. The constraints of cost and efficacy.

Decision analysis was used to examine the interrelationship of cost and success for an intervention program designed to prevent unexpected readmissions to an acute care hospital. Three separate strategies were considered: 1) intervention for patients who are at high risk for unexpected readmission; 2) intervention for all patients who are admitted to the medical service; and 3) no intervention. Risk and cost data were derived from an analysis of 4,769 patients discharged from Boston's Beth Israel Hospital medical service during a 1-year period. It was found that interventions that cost $250.00 per patient could be cost saving given reasonable expectations of success. If an intervention for high risk patients costs $250.00 per patient, overall costs would be reduced if the success rate of the intervention was 9% or greater. If the success rate exceeded 17%, offering the intervention to all patients would reduce costs. If the cost of an intervention increases by a factor of two, it must be twice as likely to succeed to achieve the same economic benefit. Decision analysis provides a useful framework for evaluation of the economic implications of intervention strategies, and it is recommended that these methods be used in the planning and implementing of medical interventions.

Ambulatory Care↗
PubMed1988

Detection and identification of transient intermediates in the reactions of tryptophan synthase with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Evidence for a tetrahedral (gem-diamine) intermediate.

The reactions of 2,3-dihydro-L-tryptophan (DHT) and oxindolyl-L-alanine (OXA) with tryptophan synthase have been investigated by rapid-scanning stopped-flow (RSSF) spectroscopy and by the concentration dependence of rates measured by single-wavelength stopped-flow (SWSF) spectroscopy. The RSSF spectral changes for DHT and OXA show the disappearance of the internal aldimine (lambda max 412 nm), the formation and decay of intermediates absorbing less than or equal to 340 nm, and the appearance of the quinonoid (lambda max 492 and 480 nm, respectively). Rate constants determined by SWSF were either well resolved (i.e., k1[DHT], k-1 greater than k2, k-2 greater than k3, k-3) or indicative of a tightly coupled system (i.e., k1[OXA], k-1 greater than or equal to k2, k-2 greater than k3, k-3). The RSSF spectral changes and SWSF kinetic studies together with computer simulations of the kinetic time courses are consistent with a mechanism that includes formation of a bleached species. Detection of these shorter wavelength species in the reactions of OXA and DHT indicates that substrate analogues with tetrahedral geometry at C-3 induce new protein-substrate interactions that result in the accumulation of species not previously detected in the tryptophan synthase system. The bleached species with lambda max less than or equal to 340 nm are proposed as the gem-diamine intermediates.

Alanine↗
PubMed1988

Mechanistic deductions from kinetic isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Erwinia herbicola and Citrobacter freundii tyrosine phenol-lyase.

The pH dependence of the kinetic parameters and primary deuterium isotope effects have been determined for tyrosine phenol-lyase from both Erwinia herbicola and Citrobacter freundii. The primary deuterium isotope effects indicate that proton abstraction from the 2-position of the substrate is partially rate-limiting for both enzymes. The C. freundii enzyme primary deuterium isotope effects [DV = 3.5 and D(V/Ktyr) = 2.5] are pH independent, indicating that tyrosine is not sticky (i.e., does not dissociate slower than it reacts to give products). Since Vmax for both tyrosine and the alternate substrate S-methyl-L-cysteine is also pH independent, substrate binds only to the correctly protonated form of the enzyme. For the E. herbicola enzyme, both Vmax and V/K for tyrosine or S-methyl-L-cysteine are pH dependent, as well as both DV and D(V/Ktyr). Thus, while both the protonated and unprotonated enzyme can bind substrate, and may be interconverted directly, only the unprotonated Michaelis complex is catalytically competent. At pH 9.5, DV = 2.5 and D(V/Ktyr) = 1.5. However, at pH 6.4 the isotope effect on both parameters is equal to 4.1. From these data, the forward commitment factor (cf = 5.2) and catalytic ratio (cvf = 1.1) for tyrosine and S-methyl-L-cysteine (cf = 2.2, cvf = 24) are calculated. Also, the Michaelis complex partition ratio (cf/cvf) for substrate and products is calculated to be 4.7 for tyrosine and 0.1 for S-methyl-L-cysteine.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗