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L M Watkins

Publications and source records attributed to L M Watkins.

9 recordsLinked to original sources
PubMed2003

Purification and characterization of the aromatic desulfinase, 2-(2'-hydroxyphenyl)benzenesulfinate desulfinase.

2-(2(')-Hydroxyphenyl)benzenesulfinate desulfinase (HPBS desulfinase) catalyzes the cleavage of the carbon-sulfur bond of 2-(2(')-hydroxyphenyl)benzenesulfinate (HPBS) to form hydroxybiphenyl and sulfite. This is the final step in the desulfurization of dibenzothiophene, the organosulfur compound used to study biodesulfurization of petroleum middle distillate. HPBS desulfinase was purified 1600-fold from Rhodococcus IGTS8. The purification was monitored using a spectrofluorimetric assay and SDS-PAGE. The pI of HPBS desulfinase is 5.6, the temperature optimum is 35 degrees C, and the pH optimum is 7.0. HPBS desulfinase has a K(m) of 0.90+/-0.15 microM and a k(cat) of 1.3+/-0.07 min(-1). Several analogs were tested for their ability to act as substrates or inhibitors of HPBS desulfinase. No alternative substrates and very few inhibitors were identified. HPBS desulfinase activity decreases in the presence of Cu(2+) and Zn(2+), while no metals significantly enhance enzyme activity. HPBS desulfinase is susceptible to tyrosine, tryptophan, and cysteine specific modification agents.

Cells, Cultured↗
PubMed2001

Orbital fractures in children.

PURPOSE: To describe the demographics, etiologic factors, clinical presentations, and outcomes of orbital fractures in children. METHODS: This was a retrospective case series of 96 consecutive patients under 18 years of age with orbital fractures presenting to the Massachusetts Eye and Ear Infirmary, including both hospitalized and nonhospitalized patients. RESULTS: Orbital fractures in children were most frequently the result of sports, assault, or motor vehicle accident. The majority of patients did not require hospitalization and were treated as outpatients. The medial wall and floor of the orbit were the most frequent locations of fracture. Approximately half of the patients in this series required surgery, most often for entrapment. There were no cases of persistent diplopia in patients in whom surgery was performed or was not indicated. Associated ocular injuries were observed in half of the patients. CONCLUSIONS: In this series of hospitalized and nonhospitalized patients, orbital fractures in children had a location pattern similar to that most frequently observed in adult patients (floor and medial wall). Orbital fractures in children frequently require surgery. The high prevalence of ocular injury in children with orbital fractures emphasizes the need for a comprehensive ophthalmic evaluation.

Adolescent↗
PubMed1998

Orbital computed tomographic characteristics of globe subluxation in thyroid orbitopathy.

OBJECTIVE: Axial globe subluxation may complicate thyroid orbitopathy. This acute event is defined by anterior displacement of the globe equator beyond the orbital rim, lid retraction behind the equator, and tethering of the optic nerve. The authors explored the factors leading to spontaneous globe subluxation in patients with thyroid orbitopathy. DESIGN: A clinical review. MAIN OUTCOME MEASURES: The medical charts and axial and coronal orbital computed tomographic (CT) scans of all the patients with spontaneous globe subluxation were reviewed. The patients were categorized according to their CT scans into type I ("lipogenic" variant) or type II ("myogenic" variant). RESULTS: Four (0.1 %) of approximately 4000 patients with thyroid orbitopathy presented with spontaneous globe subluxation. All of these patients had increased orbital fat without significant enlargement of the extraocular muscles. Thus, they represent type-I thyroid orbitopathy. CONCLUSIONS: Globe subluxation in thyroid orbitopathy requires compliance of the orbital soft tissues and extensibility of the extraocular muscles. These are characteristics of type-I orbitopathy. The increased fat content results in more compliance of the soft tissues, and the normal caliber of the muscles allows them to become more extensible. This permits the acute contraction of the eyelids posterior to the equator of the globe. Patients with type-I orbitopathy and extensive proptosis may be at relatively greater risk of having globe subluxation develop.

Acute Disease↗
PubMed1997

Augmented hydrolysis of diisopropyl fluorophosphate in engineered mutants of phosphotriesterase.

The phosphotriesterase from Pseudomonas diminuta hydrolyzes a wide variety of organophosphate insecticides and acetylcholinesterase inhibitors. The rate of hydrolysis depends on the substrate and can range from 6000 s-1 for paraoxon to 0.03 s-1 for the slower substrates such as diethylphenylphosphate. Increases in the reactivity of phosphotriesterase toward the slower substrates were attempted by the placement of a potential proton donor group at the active site. Distances from active site residues in the wild type protein to a bound substrate analog were measured, and Trp131, Phe132, and Phe306 were found to be located within 5.0 A of the oxygen atom of the leaving group. Eleven mutants were created using site-directed mutagenesis and purified to homogeneity. Phe132 and Phe306 were replaced by tyrosine and/or histidine to generate all combinations of single and double mutants at these two sites. The single mutants W131K, F306K, and F306E were also constructed. Kinetic constants were measured for all of the mutants with the substrates paraoxon, diethylphenylphosphate, acephate, and diisopropylfluorophosphate. Vmax values for the mutant enzymes with the substrate paraoxon varied from near wild type values to a 4-order of magnitude decrease for the W131K mutant. There were significant increases in the Km for paraoxon for all mutants except F132H. Vmax values measured using diethylphenylphosphate decreased for all mutants except for F132H and F132Y, whereas Km values ranged from near wild type levels to increases of 25-fold. Vmax values for acephate hydrolysis ranged from near wild type values to a 10(3)-fold decrease for W131K. Km values for acephate ranged from near wild type to a 5-fold increase. Vmax values for the mutants tested with the substrate diisopropylfluorophosphate showed an increase in all cases except for the W131K, F306K, and F306E mutants. The Vmax value for the F132H/F306H mutant was increased to 3100 s-1. These studies demonstrated for the first time that it is possible to significantly enhance the ability of the native phosphotriesterase to hydrolyze phosphorus-fluorine bonds at rates that rival the hydrolysis of paraoxon.

Aryldialkylphosphatase↗
PubMed1997

A combinatorial library for the binuclear metal center of bacterial phosphotriesterase.

Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of an extensive array of organophosphate pesticides and mammalian acetylcholinesterase nerve agents. Although the three-dimensional crystal structure of PTE has been solved (M. M. Benning et al., Biochemistry 34:7973-7978, 1995), the precise functions of the individual amino acid residues that interact directly with the substrate at the active site are largely unknown. To construct mutants of PTE with altered specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed. In this investigation, four of the six protein ligands to the binuclear metal site (His-55, His-57, His-201, and His-230) were targeted for further characterization and investigation. Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generated by simultaneously creating random combinations of histidine and cysteine codons at these four positions. The 16 possible DNA sequences were isolated and confirmed by dideoxy-DNA sequencing. The 16 mutant proteins were expressed in Escherichia coli and grown with the presence or absence of 1 mM CoCl2, ZnSO4, or CdSO4 in the growth medium. When grown in the presence of CoCl2, the H57C protein cell lysate showed greater activity for the hydrolysis of paraoxon than the wild type PTE cell lysate. H201C and H230C exhibited up to 15% of the wild-type activity, while H55C, a green protein, was inactive under all assay conditions. All other mutants had < 10(-5) of wild-type activity. None of the purified mutants that exhibited catalytic activity had a significantly altered Km for paraoxon.

Aryldialkylphosphatase↗
PubMed1997

Orbital granulocytic sarcoma in an elderly patient.

PURPOSE: To report a 71-year-old woman with acute myelogenous leukemia in remission who developed orbital granulocytic sarcoma. METHODS: The patient was referred for acute proptosis and decreased vision of the right eye. Computed tomography of the orbits demonstrated a right extraconal mass compressing the optic nerve. A right lateral orbitotomy was performed, and a portion of the mass was excised for diagnostic purposes and orbital decompression. RESULTS: Histopathologic and immunohistochemical evaluation disclosed orbital granulocytic sarcoma. With chemotherapy and radiation, vision remained stable and right proptosis resolved. CONCLUSIONS: Orbital granulocytic sarcoma is usually diagnosed in children with a history of acute myelogenous leukemia. This case demonstrated that this entity may also occur rarely in older patients with a history of acute myelogenous leukemia.

Aged↗