Enzyme catalysis and engineering.
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Biotechnology↗
Biomedical subjects
David Wilson
Publications and source records attributed to David Wilson.
Time-dependent reduction in Abeta levels after intracranial LPS administration in APP transgenic mice.
Inflammation has been argued to play a primary role in the pathogenesis of Alzheimer's disease (AD). Lipopolysaccharide (LPS) activates the innate immune system, triggering gliosis and inflammation when injected in the central nervous system. In studies described here, APP transgenic mice were injected intrahippocampally with 4 or 10 microg of LPS and evaluated 1, 3, 7, 14, or 28 days later. Abeta load was significantly reduced at 3, 7, and 14 days but surprisingly returned near baseline 28 days after the injection. No effects of LPS on congophilic amyloid deposits could be detected. LPS also activated both microglia and astrocytes in a time-dependent manner. The GFAP astrocyte reaction and the Fcgamma receptor microglial reaction peaked at 7 days after LPS injection, returning to baseline by 2 weeks postinjection. When stained for CD45, microglial activation was detected at all time points, although the morphology of these cells transitioned from an ameboid to a ramified and bushy appearance between 7 and 14 days postinjection. These results indicate that activation of brain glia can rapidly and transiently clear diffuse Abeta deposits but has no effect on compacted fibrillar amyloid.
Alzheimer Disease↗
Impact of coping and socioeconomic factors on quality of life in adults with asthma.
OBJECTIVE: The socioeconomic variables of income, race and employment status have been shown to influence health-related quality of life (HRQL) scores in persons with asthma. However, little is known about the impact of other psychological factors or perceptions of economic hardship on HRQL in asthma, despite the known influence these have on general activity levels and emotional perceptions. The aim of the present study was to assess the relationships between psychological and socioeconomic factors and HRQL and on subsequent changes to HRQL over 12 months. METHODOLOGY: Consecutive adult patients with moderate or severe asthma attending outpatient clinics, emergency departments or who were inpatients at two teaching hospitals, completed surveys of clinical status, psychological and socioeconomic variables, and HRQL instruments (SF-36 and Modified Marks Asthma Quality of Life Questionnaire [MAQLQ-M]), at baseline and over 12-months follow-up. RESULTS: Of 343 eligible subjects, survey responses were received from 293 at baseline and 232 at 12-months. Mean age was 42 years (SD 18), 67% were female, 42% had moderate, and 58% severe current asthma clinical status according to the National Asthma Education and Prevention Program guidelines criteria. In random-effects multiple regression models, after adjusting for age, gender, education, income and hospital, significant independent variables associated with each of total MAQLQ-M, SF-36 Physical Component Summary (PCS) and SF-36 Mental Component Summary (MCS) scores were avoidance coping, perceived recent financial difficulties and clinical asthma status. Additional independent associations were, for total MAQLQ-M, patient concerns about costs delaying them from seeking care (overall model r(2) = 0.69); for PCS, active coping (r(2) = 0.69) and for MCS, positive evaluations/satisfaction with illness (r(2) = 0.54). CONCLUSION: Psychological factors, particularly coping styles, need to be taken into account when considering HRQL scores as outcome measures in asthma. Interventions to improve the coping capabilities of individuals with moderate-to-severe asthma may be potentially important areas for improvement of asthma-related HRQL.
Adaptation, Psychological↗
Australian funnel-web spider venom analyzed with on-line RP-HPLC techniques.
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Animals↗
Integration of computer modeling and initial studies of site-directed mutagenesis to improve cellulase activity on Cel9A from Thermobifida fusca.
Cellulases are a complex group of enzymes that are fundamental for the degradation of amorphous and crystalline cellulose in lignocellulosic material. Unfortunately, cellulases have a low catalytic efficiency on their substrates when compared to similar enzymes such as amylases, which has led to a strong interest in improving their activities. Thermobifida fusca secretes six cellulose degrading enzymes: two exo- and three endocellulases and an endo/exocellulase Cel9A (formerly called E4). Cel9A shows unique properties because of its endo- and exocellulase characteristics, strong activity on crystalline cellulose, and good synergistic properties. Therefore, it is an excellent target for mutagenesis techniques to improve crystalline cellulose degradation. In this article, we describe research conducted to improve Cel9A catalytic efficiency using a rational design and computer modeling. A computer model of Cel9A was created using the program CHARMM plus its PDB structure and a cellohexose molecule attached to the catalytic site as a starting model. Initially molecular graphics and energy minimization were used to extend the cellulose chain to 18 glucose residues spanning the catalytic domain and cellulose-binding domain (CBD). The interaction between this cellulose chain and conserved CBD residues was determined in the model, and mutations likely to improve the binding properties of the CBD were selected. Site-directed mutations were carried out using the pET vector pET26b, Escherichia coli DH5-alpha, and the QuickChange mutagenesis method. E. coli BL21-DE3 was used for protein production and expression. The purified proteins were assayed for enzymatic activity on filter paper, swollen cellulose, bacterial microcrystalline cellulose, and carboxymethylcellulose (CMC). Mutation of the conserved residue F476 to Y476 gave a 40% improved activity in assays with soluble and amorphous cellulose such as CMC and swollen cellulose.
Actinomycetales↗
The elasticity of single titin molecules using a two-bead optical tweezers assay.
Titin is responsible for the passive elasticity of the muscle sarcomere. The mechanical properties of skeletal and cardiac muscle titin were characterized in single molecules using a novel dual optical tweezers assay. Antibody pairs were attached to beads and used to select the whole molecule, I-band, A-band, a tandem-immunoglobulin (Ig) segment, and the PEVK region. A construct from the PEVK region expressing >25% of the full-length skeletal muscle isoform was chemically conjugated to beads and similarly characterized. By elucidating the elasticity of the different regions, we showed directly for the first time, to our knowledge, that two entropic components act in series in the skeletal muscle titin I-band (confirming previous speculations), one associated with tandem-immunoglobulin domains and the other with the PEVK region, with persistence lengths of 2.9 nm and 0.76 nm, respectively (150 mM ionic strength, 22 degrees C). Novel findings were: the persistence length of the PEVK component rose (0.4-2.7 nm) with an increase in ionic strength (15-300 mM) and fell (3.0-0.3 nm) with a temperature increase (10-60 degrees C); stress-relaxation in 10-12-nm steps was observed in the PEVK construct and hysteresis in the native PEVK region. The region may not be a pure random coil, as previously thought, but contains structured elements, possibly with hydrophobic interactions.
Animals↗
Absence of BRAF and NRAS mutations in uveal melanoma.
Uveal melanoma (UM) and cutaneous melanoma (CM) differ significantly in their epidemiological, clinical, immunophenotypical, and cytogenetic features, but the molecular basis for these differences has not been delineated. CMs frequently harbor an activating mutation in either NRAS or the RAS-regulated kinase BRAF, suggesting that either of these oncogenes may increase signaling through the mitogen-activated protein (MAP) kinase pathway and promote melanoma development. The aim of this study was to examine BRAF and NRAS gene mutations in UM. Genomic DNA from CM and UM was screened for mutations in BRAF exons 11 and 15 and NRAS exons 1 and 2 using a combination of denaturing high-performance liquid chromatography and direct sequencing. Mutations in BRAF exon 15 were detected in 16 (36.4%) of 44 CMs and 0 (0%) of 62 UMs. The most common mutation in CM was V599E, but a novel point mutation (L596Q) was identified in two cases and an in-frame deletion/insertion (VKSRWK599-604D) was discovered in one case. No BRAF exon 11 mutations were observed among seven CMs and nine UMs that were wild-type for exon 15. Mutation of NRAS exon 2 was rare in CM [1 (3.7%) of 27] and absent in UM [0 (0%) of 47]. No NRAS exon 1 mutations were detected in either type of melanoma. We conclude that UMs arise independent of oncogenic BRAF and NRAS mutations, an observation that may have implications for therapies targeted to the NRAS-BRAF pathway.
Base Sequence↗
Effect of longitudinal oxygen gradients on effectiveness of manipulation of tumor oxygenation.
The purpose of this study was to test the hypothesis that longitudinal O(2) gradients in tumor affect response to manipulation of oxygenation. Previously we showed that pO(2) is higher on the fascial than the tumor surface of the R3230Ac rat mammary carcinoma when growing in a dorsal skin-fold window chamber, reflecting a longitudinal oxygen gradient. Magnetic resonance angiography verified prior results: the fascial surface has arterioles and higher vascular density than tumor; and the tumor surface has no arterioles. Phosphorescence lifetime imaging was used to measure each surface hypoxic percentage (HP; percentage of pixels < 10 mm Hg) before and after administration of mannitol or glucose (1 g/kg, i.v.) followed by O(2) breathing. The fascial surface had a smaller HP (median = 2.72%) than tumor (median = 27.94%; P = 0.0002) at baseline. HP on the fascial surface was positively correlated with HP on the tumor surface (P = 0.0067). HP decreased on the fascial surface after either sugar + O(2) (mannitol P = 0.03; glucose P = 0.06; combined P = 0.002), but HP did not change on the tumor surface. Therefore, the tumor surface is refractory to improvement in pO(2) with this method. Additional refinements may be needed to improve pO(2) of analogous regions in larger tumors; mechanism-driven suggestions are provided.
Animals↗
Helical templating of oligopeptides by cyclodextrin dimers.
beta-cyclodextrin-based receptors were synthesized and tested for their ability to induce a helical fold in peptides bearing hydrophobic amino acid residues in the i, i+11- or i, i+14-positions. Circular dichroism experiments revealed that a dimeric beta-cylodextrin receptor synthesized from a [1,1'-biphenyl]-4,4'-dithiol core demonstrated an ability to fold a designed peptide bearing the artificial amino acid L-p-t-butylphenylalanine in the i, i+11-positions, while other dimeric and monomeric receptors failed to do so. Titration studies were performed using both circular dichroism and calorimetry, the analysis of which yielded an apparent K(a) on the order of 10(4)-10(5) M(-1). However, no evidence could be obtained for helical folding with a peptide carrying tryptophan residues in place of the p-t-butylphenylalanine units. Our studies suggest that receptors of this type may be useful in molecular recognition of hydrophobic, already alpha-helical peptides in aqueous solution.
Amino Acid Sequence↗
The elasticity of single kettin molecules using a two-bead laser-tweezers assay.
Kettin is a high molecular mass protein of insect muscle associated with thin filaments and alpha-actinin in the Z-disc. It is thought to form a link between thin and thick filaments towards its C-terminus, contributing significantly to passive sarcomere stiffness. Here the elastic properties were characterised by mechanical stretches on an antibody-delimited region of the single molecule using two independent optical traps capable of exerting forces up to 150 pN. Step-like events were observed in the force-extension relationships consistent with the unfolding of Ig domains at moderate force and refolding of these domains at significantly higher forces than have been observed for related modular proteins.
Animals↗
Phosphorescence lifetime imaging in turbid media: the forward problem.
An adequate description of the propagation of near-infrared light in highly scattering media is important for imaging hidden luminescent inhomogeneities in tissues. We present an improved approach to modeling of the forward problem based on a telegraph equation. The asymptotic solutions of the telegraph equation are derived and analyzed. The expression for the mean photon path length is also obtained.
Luminescence↗
Mechanism of magnetization transfer during on-resonance water saturation. A new approach to detect mobile proteins, peptides, and lipids.
The mechanism of magnetization transfer (MT) between water and components of the proton spectrum was studied ex vivo in a perfused cell system and in vivo in the rat brain (n = 5). Water was selectively labeled and spectral buildup consequential to transfer of longitudinal magnetization was followed as a function of time. At short mixing time (T(m)), nitrogen-bound solvent-exchangeable protons were observed, predominantly assigned to amide groups of proteins and peptides. At longer T(m), intramolecular nuclear Overhauser enhancement (NOE) was observed in the aliphatic proton region, leading to a mobile-macromolecule-weighted spectrum that resembles typical protein spectra described in the literature. This effect on the proton spectrum is distinct from that of classical off-resonance MT, which has been shown to be due to the immobile solid-like proton pool. When studying a solution of major brain metabolites under physiological concentrations and conditions (pH), no transfer effects were observed, in line with expectations based on reduced NOE effects in rapidly tumbling molecules and the fast proton exchange rates of amino, amine, SH, and OH groups. The spectral intensities of the amide protons may serve as indicators for pH and cellular levels of mobile proteins and peptides, while the aliphatic components are representative of several types of mobile macromolecules, including proteins, peptides, and lipids.
Animals↗
Body mass index, waist hip ratio, and waist circumference: which measure to classify obesity?
OBJECTIVES: To determine the proportion of a representative population sample of adults in South Australia who have a body mass index (a measure of overall obesity) classified as normal or underweight, but who also have a waist circumference or waist hip ration (measures of central obesity) that indicates obesity. METHODS: A representative population sample of adults aged 18 years and over living in the north west region of Adelaide (n = 2523) were recruited to the study. Clinical measures of height, weight, waist and hip circumference were obtained and used to determine body mass index, waist hip ratio and waist circumference. RESULTS: Among women with a normal body mass index, 19.0% had a high waist circumference (> or = 80 cm) and 8.5% had a high waist hip ratio (> 0.85). Among males with a normal body mass index, 3.4% had a high waist circumference (> or = 95 cm) and 0.1% had a high waist hip ratio (> 1.0). CONCLUSIONS: Body mass index, waist hip ratio and waist circumference all have a role in the identification of those who are obese or overweight.
Adolescent↗
Population attributable risk of major depression for suicidal ideation in a random and representative community sample.
BACKGROUND: The importance of depression in suicidal behaviour and suicidal ideation is usually determined on clinical samples. However, public health planning also requires population data. This study utilised the population attributable risk statistic in determining the importance of major depression as a contributing factor to suicidal ideation in a random and representative sample of the population. METHOD: Major depression and suicidal ideation as well as demographic and clinical data were delineated in a random and representative population sample of 3010 subjects. The population attributable risk statistic was used to determine the contribution of major depression to suicidal ideation. RESULTS: Multivariate analysis demonstrated that major depression was the major contributor to the risk for suicidal ideation with a population attributable risk of 56.6%. CONCLUSIONS: These results, utilising different measures of depression and suicidal ideation to those few previous population attributable risk studies examining this issue, confirm the overwhelming importance of major depression as a contributing factor to suicidal ideation in the community.
Adolescent↗
An unusual complication of asthma--the exploding inhaler.
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Accidents, Occupational↗
The evolution of display technologies in PACS applications.
Picture archiving and communications systems (PACS) systems have been around for over a decade now. The most visible components in a PACS system are the PACS workstations. Most of the clinical users of PACS only interact with the display hardware/software pieces and never truly think about the archival and communications that occur behind the scenes. This paper discusses the evolution of PACS display technology in the past 16 yr, which can roughly be divided into three phases and will also discuss future emerging trends.
Computer Terminals↗
Self-reported patterns of health services utilisation: an urban-rural comparison in South Australia.
OBJECTIVE: To compare self-reported patterns of health service utilisation among residents of urban and rural South Australia. DESIGN, SETTING AND MAIN OUTCOME MEASURES: Secondary analysis of data generated by computer-assisted telephone interviews of 7377 adults done in 1995-6. Respondents were asked if they had used each of 18 different health services during the previous 12 months. Residence was classified in three ways: (1) capital city versus rest of the state, (2) by the Rural, Remote and Metropolitan Areas classification (RRMA) and (3) by the Accessibility and Remoteness Index for Australia classification (ARIA). RESULTS: General practitioner services were most frequently used, by approximately 89% of respondents. Only 4% reported not using any service. Comparing capital city with rest of the state, modest but statistically significant differences in utilisation (P < 0.01) were measured for nine services. In eight of these nine, utilisation was higher among rural residents. Analysing by RRMA, eight services were reportedly used differently and seven of these were the same as those identified from the capital city versus rest of state comparison. Across the five ARIA categories, six previously identified services were reported as being used differentially. Overall, rural residents had a higher than expected rate of moderate and high level of health service use. CONCLUSIONS: Self-reported use of a range of health services was broadly similar across urban and rural South Australia, with most cases of higher use were reported from rural areas rather than urban areas. Similar results were obtained when residence was classified in the three different ways.
Adult↗
Primary squamous cell carcinoma of the thyroid gland.
A case of primary squamous cell carcinoma of the thyroid is presented. Squamous epithelium is normally not present in the thyroid gland, and this tumor is exceedingly rare. Proper workup is described, including the differentiation from metastatic disease. The case reported is of a 64-year-old woman who presented with an asymptomatic nodule and underwent thyroidectomy. The final pathologic diagnosis, confirmed using immunohistochemistry for cytokeratin and transmission electron microscopy, revealed the unusual tumor. Squamous cell carcinoma of the thyroid is highly aggressive and behaves clinically like anaplastic thyroid carcinoma; the prognosis is poor. The patient described developed local recurrence and expired 7 months after resection from local invasion and airway obstruction. Unfortunately, this is the typical outcome. Chemoradiotherapy is generally not helpful, and complete resection remains the best chance for cure.
Carcinoma, Squamous Cell↗