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S H Brown

Publications and source records attributed to S H Brown.

At least 19 recordsLinked to original sources
PubMed2001

A novel carbohydrate:acceptor oxidoreductase from Microdochium nivale.

A Microdochium nivale carbohydrate:acceptor oxidoreductase was purified, cloned, heterologously expressed, and characterized. The gene encoding the protein showed one intron, and the ORF showed a sequence with low homology (< or = 25% identity or 65% similarity) to other known flavin-containing carbohydrate oxidases. The maturation of the protein required the cleavage of a tetrameric propeptide in addition to an 18 amino-acid signal peptide. The enzyme was found to have a relative molecular mass of 55 000 Da, an isoelectric point of 9, and one FAD per protein. It could oxidize mono-, oligo-, or polymeric saccharides, and transfer their electrons to O2 or other acceptors. When D-glucose served as electron-donating substrate, an activity of 2 s(-1) was observed at pH 5.5 and 23 degrees C. Among various oligosaccharides, the enzyme preferred tetrameric dextrins, indicating a favorable interaction of four linked glucose units with the substrate pocket. The unique structure and ability of oxidizing oligo/polymeric saccharides suggest a promising prospect of this enzyme for various industrial/medicinal applications.

Alcohol Oxidoreductases↗
PubMed2000

Using DNA-tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae.

Using DNA-tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae. Fungal Genetics and Biology 29, 28-37. Restriction enzyme-mediated integration (REMI) has been employed as a mutagen to generate two insertion libraries in an Aspergillus oryzae strain expressing a Thermomyces lanuginosus lipase. The REMI libraries were created using linearized plasmid containing the A. oryzae pyrG and either BamHI or EcoRI enzyme. The libraries were screened for lipase production, and mutants with increased production were isolated. The genomic DNA flanking the integration event was cloned from one of the mutants with increased lipase titers (DEBY10.3). Nucleotide sequence of the flanking DNA revealed similarity to the Aspergillus nidulans palB gene. Disruption of the palB gene in a strain producing lipase resulted in increased lipase expression. Additionally, complementation of the palB phenotype of DEBY10.3 led to a decrease in lipase production. These lines of evidence demonstrate that the increase in lipase yield in DEBY10.3 is linked to the palB phenotype generated by the integration of the pyrG gene into the palB gene. The results also demonstrated that tagged mutagenesis with REMI can be used to identify genes that influence expression of heterologous proteins.

Aspergillus oryzae↗
PubMed2000

Motor control testing of upper limb function after botulinum toxin injection: a case study.

OBJECTIVE: To evaluate changes in upper extremity function in a hemiparetic patient after treatment with botulinum toxin (BTX) using motor-control testing (MCT) techniques. DESIGN: Interventional with longitudinal study, open label. SETTING: A children's hospital and a motor-control laboratory at a major academic center. PARTICIPANTS: A 16-year-old male with right hemiparetic cerebral palsy and a healthy 12-year-old control subject. INTERVENTIONS: BTX injections to the elbow and wrist flexors. MAIN OUTCOME MEASURES: MCT was used to examine 4 upper extremity movements: forward reach, bilateral rhythmic movements (both muscle homologous and direction homologous), isometric pinch, and hand tapping. The patient was tested before treatment and at 2, 4, 6, 12, 18, and 24 weeks after treatment. In addition, range of motion (ROM), the Ashworth scale of spasticity, Functional Independence Measure, and the mobility and activities of daily living (ADL) sections of the Pediatric Evaluation of the Disability Inventory were performed. RESULTS: Forward reach demonstrated little change initially despite patient reports of "feeling looser." Improvement was noted after 18 weeks, but returned to baseline level at 24 weeks. Bilateral rhythmic movements also showed slight improvement at 18 weeks. Pinch force increased significantly after 2 weeks, but declined again at 6 weeks. Improvements occurred in ROM and the Ashworth rating of spasticity, but were not temporally associated with each other or with MCT results. Functional assessment data did not change during the study period. CONCLUSIONS: Improvements in more complex motor tasks were noted after significant delay from the time of treatment, while simpler tasks demonstrated a more rapid improvement, followed by a rapid return to baseline levels. This case suggests that MCT techniques can provide quantitative and qualitative data, which can add new information about upper extremity motor disability and the outcome of treatment.

Adolescent↗
PubMed1999

Empirical derivation of an electronic clinically useful problem statement system.

Problem lists are tools to improve patient management. In the medical record, they connect diagnoses to therapy, prognosis, and psychosocial issues. Computer-based problem lists enhance paper-based approaches by enabling cost-containment and quality assurance applications, but they require clinically expressive controlled vocabularies. Because existing controlled vocabularies do not represent problem statements at a clinically useful level, we derived a new canonical problem statement vocabulary through semi-automated analysis and distillation of provider-entered problem lists collected over 6 years from 74,696 patients. We combined automated and manual methods to condense 891,770 problem statements entered by 1961 care providers at Grady Memorial Hospital in Atlanta, Georgia, to 15,534 Canonical Clinical Problem Statement System (CCPSS) terms. The nature and frequency of problem statements were characterized, interrelations among them were enumerated, and a database capturing the epidemiology of problems was created. The authors identified 23,503 problem relations (co-occurrences, sign-symptom complexes, and differential diagnoses) and 22,690 modifier words that further categorized "canonical" problems. To assess completeness, CCPSS content was compared with that of the 1997 Unified Medical Language System Metathesaurus (containing terms from 44 clinical vocabularies). Unified Medical Language System terms expressed 25% of individual CCPSS terms exactly (71% of problems by frequency), 27% partially, and 48% poorly or not at all. Clinicians judged that CCPSS terms completely captured their clinical intent for 84% of 686 randomly selected free-text problem statements. The CCPSS represents clinical concepts at a level exceeding that of previous approaches. A similar national approach could create a standardized, useful, shared resource for clinical practice.

Humans↗
PubMed1999

Targeted mutations in a Trametes villosa laccase. Axial perturbations of the T1 copper.

Trametes villosa laccase was mutated on a tetrapeptide segment near the type 1 site. The mutations F463M and F463L were at the position corresponding to the type 1 copper axial methionine (M517) ligand in Zucchini ascorbate oxidase. The mutations E460S and A461E were near the T1 copper site. The mutated Trametes laccases were expressed in an Aspergillus oryzae host and characterized. The E460S mutation failed to produce a transformant with meaningful expression. The F463L and A461E mutations did not significantly alter the molecular and enzymological properties of the laccase. In contrast, the F463M mutation resulted in a type 1 copper site with an EPR signal intermediate between that of the wild type laccase and plastocyanin, an altered UV-visible spectrum, and a decreased redox potential (by 0.1 V). In oxidizing phenolic substrate, the mutation led to a more basic optimal pH as well as an increase in kcat and Km. These effects are attributed to a significant perturbation of the T1 copper center caused by the coordination of the axial methionine (M463) ligand.

Base Sequence↗
PubMed1999

Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase lcc1.

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K(m) of 21 +/- 2 microM and a catalytic constant of 200 +/- 10 min(-1) for O(2) with 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.

Amino Acid Sequence↗
PubMed1999

No free lunch: institutional preparations for computer-based patient records.

The Veterans Administration (VA) is aggressively pursuing computer-based medical records by deploying the Computerized Patient Record System (CPRS) across its 150 medical centers and 400 outpatient clinics. CPRS's client-server, patient-centered approach to clinical computing is a departure from VA's traditional terminal-compatible, department-centered approach. Although the CPRS software is freely distributed, institutional readiness for computer-based patient records has proven expensive. Preparations include organizational changes, human resource development, hardware deployment, physical plant upgrades, and software testing. This paper details CPRS preparations and their costs at one VA Medical Center. Lessons learned during the process are summarized.

Computer Systems↗
PubMed1998

Site-directed mutations in fungal laccase: effect on redox potential, activity and pH profile.

A Myceliophthora thermophila laccase and a Rhizoctonia solani laccase were mutated on a pentapeptide segment believed to be near the type-1 Cu site. The mutation L513F in Myceliophthora laccase and the mutation L470F in Rhizoctonia laccase took place at a position corresponding to the type-1 Cu axial methionine (M517) ligand in Zucchini ascorbate oxidase. The triple mutations V509L,S510E,G511A in Myceliophthora laccase and L466V,E467S,A468G in Rhizoctonia laccase involved a sequence segment whose homologue in ascorbate oxidase is flanked by the M517 and a type-1 Cu-ligating histidine (H512). The single mutation did not yield significant changes in the enzymic properties (including any significant increase in the redox potential of the type-1 Cu). In contrast, the triple mutation resulted in several significant changes. In comparison with the wild type, the Rhizoctonia and Myceliophthora laccase triple mutants had a phenol-oxidase activity whose pH optimum shifted 1 unit lower and higher, respectively. Although the redox potentials were not significantly altered, the Km, kcat and fluoride inhibition of the laccases were greatly changed by the mutations. The observed effects are interpreted as possible mutation-induced structural perturbations on the molecular recognition between the reducing substrate and laccase and on the electron transfer from the substrate to the type-1 Cu centre.

Amino Acid Sequence↗
PubMed1998

Crystal structure of the type-2 Cu depleted laccase from Coprinus cinereus at 2.2 A resolution.

Laccase catalyses the oxidation of a variety of organic substrates coupled to the reduction of oxygen to water. It is widely believed to be the simplest representative of the ubiquitous blue multi-copper oxidase family. Laccase is implicated in a wide spectrum of biological activities and, in particular, plays a key role in morphogenesis, development and lignin metabolism in fungi and plants. The structure of laccase from the fungus Coprinus cinereus has been determined by X-ray crystallography at a resolution of 2.2 A. Laccase is a monomer composed of three cupredoxin-like beta-sandwich domains, similar to that found in ascorbate oxidase. In contrast to ascorbate oxidase, however, the mononuclear type-1 Cu site lacks the axial methionine ligand and so exhibits trigonal planar coordination, consistent with its elevated redox potential. Crucially, the structure is trapped in a Cu depleted form in which the putative type-2 Cu atom is completely absent, but in which the remaining type-1 and type-3 Cu sites display full occupancy. Type-2 Cu depletion has unexpected consequences for the coordination of the remaining type-3 Cu atoms.

Amino Acid Sequence↗
PubMed1997

Quantification of upper extremity function using kinematic analysis.

OBJECTIVE: To illustrate the applicability of motor control analytical techniques to the assessment of upper limb dysfunction in children with ataxia. DESIGN: Descriptive case series. SETTING: The study sample was selected from an outpatient pediatric rehabilitation clinic and testing was performed in a research laboratory. PARTICIPANTS: Four children with upper limb ataxia and seven healthy children were examined. All subjects were recruited on a volunteer basis. Criteria for inclusion (ataxic group) included: (1) age 6 to 15 yrs; (2) ambulatory with assistive devices. MAIN OUTCOME MEASURES: Quantitative measures of elbow kinematics (movement speed and duration) and spatio-temporal "portraits" of elbow movement during unilateral and bilateral forward reaching movements. RESULTS: Movements made by ataxic subjects were characterized by lower peak velocities, prolonged durations, and increased variability compared with normal subjects. In the one subject with unilateral ataxia, interlimb coordination was severely disrupted during the performance of coupled, bilateral arm movements. In addition to changes in specific kinematic values (eg, peak velocity), phase plane and angle-angle displacement curves revealed marked spatio-temporal variability throughout the movement, the magnitude of which was correlated with severity of ataxia. CONCLUSION: The application of the quantitative motor control methods described in this report can provide rehabilitation specialists with a simple yet sensitive means to evaluate treatment and progression of a wide variety of motor disorder conditions. These techniques are particularly well suited to pediatric populations as young as 6 years.

Adolescent↗
PubMed1997

Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae.

A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined. The deduced amino acid sequence of M. thermophila laccase (MtL) shows homology to laccases from diverse fungal genera. A vector containing the M. thermophila laccase coding region, under transcriptional control of an Aspergillus oryzae alpha-amylase gene promoter and terminator, was constructed for heterologous expression in A. oryzae. The recombinant laccase expressed in A. oryzae was purified to electrophoretic homogeneity by anion-exchange chromatography. Amino-terminal sequence data suggests that MtL is synthesized as a preproenzyme. The molecular mass was estimated to be approximately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that MtL contains 40 to 60% glycosylation. The laccase shows an absorbance spectrum that is typical of blue copper oxidases, with maxima at 276 and 589 nm, and contains 3.9 copper atoms per subunit. With syringaldazine as a substrate, MtL has optimal activity at pH 6.5 and retains nearly 100% of its activity when incubated at 60 degrees C for 20 min. This is the first report of the cloning and heterologous expression of a thermostable laccase.

Aspergillus oryzae↗
PubMed1997

Rhythmic auditory-motor facilitation of gait patterns in patients with Parkinson's disease.

OBJECTIVES: The effect of rhythmic auditory stimulation (RAS) on gait velocity, cadence, stride length, and symmetry was studied in 31 patients with idiopathic Parkinson's disease, 21 of them on (ON) and 10 off medication (OFF), and 10 healthy elderly subjects. METHOD: Patients walked under four conditions: (1) their own maximal speed without external rhythm; (2) with the RAS beat frequency matching the baseline cadence; (3) with RAS 10% faster than the baseline cadence; (4) without rhythm to check for carry over from RAS. Gait data were recorded via a computerised foot switch system. The RAS was delivered via a 50 ms square wave tone embedded in instrumental music (Renaissance style) in 2/4 metre prerecorded digitally on a sequencer for variable tempo reproduction. Patients on medication were tested in the morning 60-90 minutes after medication. Patients off medication were tested at the same time of day 24 hours after the last dose. Healthy elderly subjects were tested during the same time of day. RESULTS: Faster RAS produced significant improvement (P < 0.05) in mean gait velocity, cadence, and stride length in all groups. Close synchronisation between rhythm and step frequency in the controls and both Parkinson's disease groups suggest evidence for rhythmic entrainment mechanisms even in the presence of basal ganglia dysfunction. CONCLUSIONS: The results are consistent with and extend prior reports of rhythmic auditory facilitation in Parkinson's disease gait when there is mild to moderate impairment, and suggest a technique for gait rehabilitation in Parkinson's disease.

Acoustic Stimulation↗
PubMed1997

Cross-institutional reuse of a problem statement knowledge base.

This article describes client and server applications for a problem statement knowledge base derived from a large corpus of provider entered terminology. The current status and potential for integration of the server into the Vanderbilt University Medical Center computing environment are discussed. Finally, an experiment in multiple dimensions of reuse for problem list terms is introduced, and possible strategies to mediate between free text and coded data are examined.

Artificial Intelligence↗
PubMed1996

A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability.

A series of fungal laccases (Polyporus pinsitus, Rhizoctonia solani, Myceliophthora thermophila, Scytalidium thermophilum) and one bilirubin oxidase (Myrothecium verrucaria) have been studied to determine their redox potential, specificity, and stability. Polyporus and Rhizoctonia laccases possess potentials near 0.7-0.8 V (vs. NHE), while other oxidases have potentials near 0.5 V. It is observed that higher redox potential correlates with higher activity. By EPR, no significant change in the geometry of type 1 copper (II) site is observed over this series. At the optimal pH, the two substrates studied, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine, show Km values ranging form 10 to 120 and from 1 to 45 microM; and kcat values ranging from 50 to 16 000 and 200 to 3000 per min, respectively. The enzymes are more stable in the neutral-alkaline pH range. The thermal stability is in the order of bilirubin oxidase equivalent to Myceliophthora laccase equivalent to Scytalidium laccase > Polyporus laccase > Rhizoctonia laccase. Based on these results and the sequence alignments made against Zucchini ascorbate oxidase it is speculated that structural differences in the substrate-activation site (a 'blue', type 1 copper center) control the redox potential range as well as substrate specificity, and the cystine content contributes to stability.

Electron Spin Resonance Spectroscopy↗
PubMed1996

The identification and characterization of four laccases from the plant pathogenic fungus Rhizoctonia solani.

Four distinct laccase genes, lcc1, lcc2, lcc3 and lcc4, have been identified in the fungus Rhizoctonia solani. Both cDNA and genomic copies of these genes were isolated and characterized. Hybridization analyses indicate that each of the four laccase genes is present in a single copy in the genome. The R. solani laccases can be divided into two groups based on their protein size, intron/exon organization, and transcriptional regulation. Three of these enzymes have been expressed in the fungus Aspergillus oryzae. Two of the recombinant laccases, r-lcc1 and r-lcc4, as well as the native lcc4 enzyme were purified and characterized. The purified proteins are homodimeric, comprised of two subunits of approximately 66kDa for lcc4 and 50-100kDa for the recombinant lcc1 protein. These laccases have spectral properties that are consistent with other blue copper proteins. With syringaldazine as a substrate, lcc4 has optimal activity at pH7, whereas lcc1 has optimal activity at pH6.

Amino Acid Sequence↗
PubMed1996

Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.

Amino Acid Sequence↗
PubMed1996

Basal ganglia and cerebellar impairment differentially affect the amplitude and time scaling during the performance of forearm step tracking movements.

Forearm step tracking movements of different amplitudes were analysed in nine patients with cerebellar and five patients with parkinsonian symptoms in comparison to six normal subjects. Movements were made under two instructions: 1) track "as fast as possible" and 2) "as precisely as possible". Movement duration, acceleration duration and peak velocity increased with increasing amplitude. The ratio between acceleration and deceleration duration was independent of movement amplitude in the parkinsonian patients and the normal subjects. In the cerebellar patients, however, this ratio decreased with increasing amplitude. This impairment of the relative timing between acceleration and deceleration duration implies that, in contrast to parkinsonian patients, cerebellar patients cannot anymore utilize physiological scaling mechanisms when performing movements of different amplitudes.

Adult↗