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Biomedical subjects

L L Wong

Publications and source records attributed to L L Wong.

At least 37 records · Page 2Linked to original sources
PubMed1999

The thermodynamics and kinetics of electron transfer in the cytochrome P450cam enzyme system.

In anaerobic environments the first electron transfer in substrate-free P450cam is known to be thermodynamically unfavourable, but in the presence of dioxygen the reduction potential for the reaction shifts positively to make electron transfer thermodynamically favourable. Nevertheless a slower rate of electron transfer is observed in the substrate-free P450cam compared to substrate-bound P450cam. The ferric haem centre in substrate-free P450cam changes from six co-ordinate to five co-ordinate when reduced whereas in substrate-bound P450cam the iron centre remains five co-ordinate in both oxidation states. The slower rate of electron transfer in the substrate-free P450cam is therefore attributed to a larger reorganisation energy as predicted by Marcus theory.

Animals↗
PubMed1999

Surface-modified mutants of cytochrome P450cam: enzymatic properties and electrochemistry.

We report the electrochemistry of genetic variants of the haem monooxygenase cytochrome P450cam. A surface cysteine-free mutant (abbreviated as SCF) was prepared in which the five surface cysteine residues Cys-58, Cys-85, Cys-136, Cys-148 and Cys-334 were changed to alanines. Four single surface cysteine mutants with an additional mutation, R72C, R112C, K344C or R364C, were also prepared. The haem spin-state equilibria, NADH turnover rates and camphor-hydroxylation properties, as well as the electrochemistry of these mutants are reported. The coupling of a redox-active label, N-ferrocenylmaleimide, to the single surface cysteine mutant SCF-K344C, and the electrochemistry of this modified mutant are also described.

Animals↗
PubMed1999

Mutations of glutamate-84 at the putative potassium-binding site affect camphor binding and oxidation by cytochrome p450cam.

Cytochrome P450cam (CYP101) from Pseudomonas putida is unusual among P450 enzymes in that it exhibits co-operative binding between the substrate camphor and a potassium ion. This behaviour has been investigated by mutagenesis of Glu84, a surface residue which forms part of the cation-binding site. Substitutions that neutralize or reverse the charge of this side chain are shown to disrupt the co-operativity of potassium and camphor binding by P450cam, and also to influence the catalytic activity. In particular, replacement of Glu84 by positively charged residues such as lysine results in increased high-spin haem fractions and camphor turnover activities in the absence of potassium, along with decreased camphor dissociation constants. However, in the presence of potassium the camphor dissociation constants of these mutants are significantly increased compared with the wild-type, although the camphor turnover activities remain marginally higher. In contrast, substitution by aspartate results in tighter binding of both potassium and camphor, but has little effect on the enzymatic activity. In all cases the reaction remains essentially 100% coupled and gives 5-exo-hydroxycamphor as the only product. These results suggest that an anionic side chain at the 84 position is crucial for the co-operativity of camphor and cation binding, and that the physiological role for potassium binding by cytochrome P450cam is to promote camphor binding even at the expense of turnover rate, thus allowing the organism to utilize low environmental concentrations of this substrate for growth.

Amino Acid Substitution↗
PubMed1999

Diagnosis of dorsal interosseous pseudotumours by magnetic resonance imaging.

Two middle-aged-patients each presenting with a progressively enlarging mass in the first dorsal web space of their hands are reported. Magnetic resonance imaging (MRI) demonstrated the cause to be a hypertrophic first dorsal interosseous muscle, with normal T1, T2 and post-gadopentetate dimeglumine signal characteristics. The ability of MRI to diagnose anatomical variants of hand muscles is important in the clinical management of patients with these pseudotumours.

Adult↗
PubMed1999

Comparison of performance with wide dynamic range compression and linear amplification.

This study compared subject performance and preference using a compression-limiting hearing aid set to linear amplification (program 1) and wide dynamic range compression (WDRC, program 2). The frequency responses of the hearing aid were matched to a 65 dB SPL signal and maximum output to a 90 dB SPL signal. Twenty subjects with moderate to moderately severe sensorineural hearing loss were tested. Speech recognition scores and speech reception thresholds were obtained both in quiet and in noise. Subjective preference for WDRC or linear amplification was measured via a paired-comparison procedure on "loudness appropriateness," "clarity," and "pleasantness" to continuous discourse presented in quiet and in noise. Results suggested that WDRC yielded better speech intelligibility in quiet for low-level signals and no difference in speech intelligibility in noise compared to linear amplification. Subjects preferred WDRC for loudness to both high- and low-level signals and for pleasantness to high-level signals.

Adolescent↗
PubMed1998

An improved procedure for the preparation of X-ray diffraction-quality crystals of cytochrome p450cam.

A procedure for the crystallization of recombinant cytochrome P450cam has been developed which avoids the difficulties inherent in the glass-capillary free-interface diffusion method reported previously. The surface mutation, Cys334-->Ala (C334A), originally designed to prevent dimer formation and thus improve routine handling of the enzyme, facilitates crystallization by the hanging-drop vapour-diffusion technique. Crystals of (C334A)P450cam grow within 48 h and diffract to beyond 1.2 A at 100 K in-house on a Siemens multiwire area detector. Data have been collected from the camphor-bound form to 1.35 A.

Alanine↗
PubMed1998

The oxidation of naphthalene and pyrene by cytochrome P450cam.

Mutants of the heme monooxygenase cytochrome P450cam in which Y96 had been replaced with hydrophobic residues, have been shown to oxidise naphthalene and pyrene with rates one to two orders of magnitude faster than the wild-type. Naphthalene was oxidised to 1- and 2-naphthol, probably via the 1,2-oxide intermediate. In the case of the Y96F mutant, naphthalene was oxidised at a rate comparable to camphor. Pyrene oxidation gave 1,6- and 1,8-pyrenequinone with no evidence for attack at the K-region, in contrast to mammalian enzymes. The results show that the Y96 residue plays a key role in controlling the substrate range of P450cam.

Camphor 5-Monooxygenase↗
PubMed1998

Trendelenburg positioning and continuous lateral rotation improve oxygenation in hepatopulmonary syndrome after liver transplantation.

Hepatopulmonary syndrome (HPS) is characterized by hypoxia, orthodeoxia, and platypnea, associated with severe chronic liver disease. Liver transplantation is generally viewed as the only curative treatment for this syndrome, but it may be complicated by prolonged hypoxia after the procedure. We report on a 58-year-old female patient with alcoholic cirrhosis and HPS who underwent liver transplantation. She developed severe hypoxia after transplantation that improved with the initiation of Trendelenburg's positioning in combination with continuous lateral rotation. Although many techniques for dealing with posttransplant hypoxia for HPS have been described, positioning is a simple maneuver that may correct the pathophysiologic abnormalities seen in HPS by gravitationally shifting blood away from the lung bases to improve oxygenation. Although this represents a single patient, the results were reproducible, and the intervention is simple and associated with minimal potential complications. The authors think this is a useful intervention to apply to the severely hypoxic patient with HPS, and a trial with more patients is warranted.

Female↗
PubMed1998

Cytochrome P450 monooxygenases.

The field of P450 research is progressing rapidly. The active sites of structurally characterized bacterial P450 enzymes have been rationally re-engineered to alter both substrate specificity and selectivity of substrate oxidation. Many human P450 enzymes have been functionally expressed and new methods of substrate turnover described. These developments, arising from our increased understanding of the chemistry and molecular biology of P450 enzymes, open up new and exciting research directions.

Bacterial Proteins↗
PubMed1997

The cost of dying of end-stage liver disease.

BACKGROUND: The high cost of liver transplantation is well known. The cost of dying of complications of end-stage liver disease (ESLD) without transplant, however, has not been well documented. METHODS: For a 5-year period (1991-1995), in 153 patients, mean inpatient hospital charges and length of stay were analyzed in 6 groups of patients: (1) patients admitted with the primary diagnosis of esophageal varices, (1a) the subset of group 1 patients who died on this admission, (2) patients admitted to the liver team who died of complications from ESLD, (3) patients who underwent transjugular intrahepatic portosystemic shunts, (4) patients who underwent surgical shunt for bleeding varices, and (5) patients who underwent liver transplantation. RESULTS: One hundred twenty-nine patients with esophageal varices were hospitalized 13.7 days with a mean charge of $30,980 for each of 202 admissions. Of these, 38 died after 24 days with a mean charge of $67,091. Seven patients admitted to the liver team died of complications of ESLD at $110,576 per admission. Transjugular intrahepatic portosystemic shunt was performed in 17 patients with a mean charge of $43,209. Six patients underwent surgical shunt for $53,994. Mean charge for 7 liver transplantations was $222,968. During the study period, 36.7% of all charges were for patients who died. CONCLUSIONS: It is difficult to estimate the total cost of ESLD; however, in evaluating inpatient costs, we see that it is expensive and significant amounts are spent on patients who die. Further study is necessary to determine which factors can optimize the cost of ESLD.

Adult↗
PubMed1997

The catalytic activity of cytochrome P450cam towards styrene oxidation is increased by site-specific mutagenesis.

The styrene oxidation activity of cytochrome P450cam, has been greatly improved by rational protein engineering. Compared to the wild-type enzyme, the active-site mutants Y96A and Y96F bound styrene more tightly, consumed NADH more rapidly, and were more efficient at utilising reducing equivalents for product formation. Styrene oxide formation rates were enhanced 9-fold in the Y96A mutant relative to wild-type, and 25-fold in the Y96F mutant, thus demonstrating the effectiveness of active-site redesign in improving the activity of a haem monooxygenase towards an unnatural substrate.

Binding Sites↗
PubMed1997

Calciphylaxis in patients on hemodialysis: a prevalence study.

BACKGROUND: Calciphylaxis is characterized by painful, violaceous, mottled skin lesions (livedo reticularis) that may progress to tissue necrosis, nonhealing ulcers, gangrene, and potentially amputation, sepsis, or death. The prevalence and characteristics of patients who have calciphylaxis need further identification to predict which patients on dialysis may benefit from close monitoring or early surgical intervention. METHODS: All 242 patients undergoing hemodialysis in an outpatient unit were reviewed retrospectively during a 15-month cross-sectional study of the prevalence and characteristics of calciphylaxis. RESULTS: Ten patients (prevalence, 4.1%) had calciphylaxis. Patients with calciphylaxis were significantly younger (49 versus 60 years; p = 0.01), had undergone hemodialysis longer (80 versus 20 months; p < 0.0001), and had higher median serum calcium (9.7 versus 9.2 mg/dl; p = 0.03), phosphate (8.2 versus 5.7 mg/dl; p = 0.001), calcium phosphate product (81.5 versus 52.9; p = 0.0004), parathyroid hormone (1496 versus 138 pg/ml; p < 0.0001), and alkaline phosphatase levels (188 versus 89 IU/L; p = 0.0001). Bone surveys were positive in all 10 patients with calciphylaxis compared with 49 (21%) of the 232 patients without calciphylaxis (p < 0.0001). All patients who underwent parathyroidectomy for calciphylaxis had dramatic healing of the ulcers. CONCLUSIONS: The presence of calciphylaxis is higher among younger patients who had undergone longer periods of hemodialysis. Therefore this group of patients should be monitored aggressively and treated expeditiously for complications of secondary hyperparathyroidism.

Adult↗
PubMed1997

The dimerization of Pseudomonas putida cytochrome P450cam: practical consequences and engineering of a monomeric enzyme.

Cytochrome P450cam dimerizes via the formation of an intermolecular disulfide bond, complicating the storage and handling of the enzyme, particularly at higher concentrations. The dimeric enzyme is 14% less active than the monomer and forms at a slow but significant rate even at 4 degrees C [k = 1.09 x 10(-3) mM(-1) h(-1)]. To eliminate any ambiguity introduced by dimer formation and to simplify handling and storage of the enzyme, site-directed mutagenesis was used to identify C334 as the single cysteine residue responsible for the formation of the disulfide linkage and to engineer a monomeric enzyme by substituting an alanine in its place. The C334A mutant is identical with the wild-type P450cam monomer in terms of optical spectra, camphor binding and turnover activity, but shows no evidence of dimerization and aggregation even at millimolar concentrations. Preliminary 1H NMR investigations also indicate a significant improvement in the quality of spectra obtained with this mutant. (C334A)P450cam is therefore proposed as an alternative to the wild-type enzyme-a base mutant otherwise identical with the wild-type but with improved handling characteristics.

Camphor↗
PubMed1997

Open heart surgery in patients with end-stage renal disease.

This case-control study evaluated the mortality and morbidity in patients with end-stage renal disease (ESRD) who underwent open heart surgery, as compared with matched control patients. Outcome measures included length of use of ventilators, vasopressor agents, number of blood units transfused, chest tube output, and length of stay. Hospital length of stay was longer in patients with renal failure, but overall morbidity and mortality were not statistically different. Conclusions include that elective open heart surgery can be performed safely in ESRD patients using routine perioperative management.

Aged↗
PubMed1997

Clinics in diagnostic imaging (22). Calcified peritoneal carcinomatosis.

A 37-year-old woman, with previously resected mucinous adenocarcinoma of the colon, presented with menorrhagia and a palpable pelvic mass. Radiographs and computed tomography (CT) demonstrated calcified peritoneal carcinomatosis in the abdomen and pelvis. Types of abdominal calcifications and imaging of intraperitoneal masses, particularly of peritoneal tumours, are discussed.

Adenocarcinoma, Mucinous↗
PubMed1996

N-(2-ferrocene-ethyl)maleimide: a new electroactive sulphydryl-specific reagent for cysteine-containing peptides and proteins.

We report the synthesis and application of a specific electroactive label, N-(2-ferrocene-ethyl)maleimide, which provides new redox properties to organic compounds and proteins possessing sulphydryl groups. Its reaction conditions with the cysteine-containing peptide, glutathione, and a terminal monooxygenase enzyme, cytochrome P450cam are presented. The labelled peptide and enzyme acquired reversible electrochemical properties due to the attached ferrocene moiety.

Bacillus cereus↗