The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13.

Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicomponent aromatic ring monooxygenase made up of three moieties: a reductase (PHR), an oxygenase (PHO) and a regulative component (PHI). The function of the oxygenase component (PHO), here characterized for the first time, is to bind molecular oxygen and catalyse the mono-hydroxylation of substrates (phenol, and with less efficiency, chloro- and methyl-phenol and naphthol). PHO was purified from extracts of A. radioresistens S13 cells and shown to be a dimer of 206 kDa. Each monomer is composed by three subunits: alpha (54 kDa), beta (38 kDa) and gamma (11 kDa). The gene encoding PHO alpha (named mopN) was cloned and sequenced and the corresponding amino acid sequence matched with that of functionally related oxygenases. By structural alignment with the catalytic subunits of methane monooxygenase (MMO) and alkene monooxygenase, we propose that PHO alpha contains the enzyme active site, harbouring a dinuclear iron centre Fe-O-Fe, as also suggested by spectral analysis. Conserved hydrophobic amino acids known to define the substrate recognition pocket, are also present in the alpha-subunit. The prevalence of alpha-helices (99.6%) as studied by CD confirmed the hypothized structural homologies between PHO and MMO. Three parameters (optimum ionic strength, temperature and pH) that affect kinetics of the overall phenol hydroxylase reaction were further analyzed with a fixed optimal PHR/PHI/PHO ratio of 2/1/1. The highest level of activity was evaluated between 0.075 and 0.1 m of ionic strength, the temperature dependence showed a maximum of activity at 24 degrees C and finally the pH for optimal activity was determined to be 7.5.     

Psychometric Properties of the Problem Areas in Diabetes (PAID) Instrument in Singapore

Background

Emotional distress is an important dimension in diabetes, and several instruments have been developed to measure this aspect. The Problem Areas in Diabetes (PAID) scale is one such instrument which has demonstrated validity and reliability in Western populations, but its psychometric properties in Asian populations have not been examined.

Methods

This was a secondary analysis of data from patients with Type 2 diabetes mellitus recruited through convenience sampling from a diabetes specialist outpatient clinic in Singapore. The following psychometric properties were assessed: Construct validity through confirmatory factor analysis (CFA) and Rasch analysis, concurrent validity through correlation with related scales (Kessler Psychological Distress Scale, Diabetes Health Profile—psychological distress, Audit of Diabetes Dependent Quality of Life), reliability through assessment of internal consistency and floor and ceiling effects, and sensitivity by estimating effect sizes for known clinical and social functioning groups.

Results

203 patients with mean age of 45±12 years were analysed. None of the previously published model structures achieved a good fit on CFA. On Rasch analysis, four items showed poor fit and were removed. The abridged 16-item PAID mapped to a single latent trait, with a high degree of internal consistency (Cronbach ɑ 0.95), but significant floor effect (24.6% scoring at floor). Both 20-item and 16-item PAID scores were moderately correlated with scores of related scales, and sensitive to differences in clinical and social functioning groups, with large effect sizes for glycemic control and diabetes related complications, nephropathy and neuropathy.

Conclusion

The abridged 16-item PAID measures a single latent trait of emotional distress due to diabetes whereas the 20-item PAID appears to measures more than one latent trait. However, both the 16-item and 20-item PAID versions are valid, reliable and sensitive for use among Singaporean patients with diabetes.     

Phenol hydroxylase from Acinetobacter radioresistens is a multicomponent enzyme. Purification and characterization of the reductase moiety.

This paper reports the isolation and characterization of phenol hydroxylase (PH) from a strain belonging to the Acinetobacter genus. An Acinetobacter radioresistens culture, grown on phenol as the only carbon and energy source, produced a multicomponent enzyme system, located in the cytoplasm and inducible by the substrate, that is responsible for phenol conversion into catechol. Because of the wide diffusion of phenol as a contaminant, the present work represents an initial step towards the biotechnological treatment of waste waters containing phenol. The reductase component of this PH system has been purified and isolated in large amounts as a single electrophoretic band. The protein contains a flavin cofactor (FAD) and an iron-sulfur cluster of the type [2Fe-2S]. The function of this reductase is to transfer reducing equivalents from NAD(P)H to the oxygenase component. In vitro, the electron acceptors can be cytochrome c as well as other molecules such as 2, 6-dichlorophenolindophenol, potassium ferricyanide, and Nitro Blue tetrazolium. The molecular mass of the reductase was determined to be 41 kDa by SDS/PAGE and 38.8 kDa by gel permeation; its isoelectric point is 5.8. The N-terminal sequence is similar to those of the reductases from A. calcoaceticus NCIB 8250 (10/12 identity) and Pseudomonas CF600 (8/12 identity) PHs, but much less similar (2/12 identity) to that of benzoate dioxygenase reductase from A. calcoaceticus BD413. Similarly, the internal peptide sequence of the A. radioresistens PH reductase displays a good level of identity (9/10) with both A. calcoaceticus NCIB 8250 and Pseudomonas CF600 PH reductase internal peptide sequences but a poorer similarity (3/10) to the internal peptide sequence of benzoate dioxygenase reductase from A. calcoaceticus BD413.     

Determinants of Health-Related Quality of Life (HRQoL) in the Multiethnic Singapore Population – A National Cohort Study

Background

HRQoL is an important outcome to guide and promote healthcare. Clinical and socioeconomic factors may influence HRQoL according to ethnicity.

Methodology

A multiethnic cross-sectional national cohort (N = 7198) of the Singapore general population consisting of Chinese (N = 4873), Malay (N = 1167) and Indian (N = 1158) adults were evaluated using measures of HRQoL (SF-36 version 2), family functioning, health behaviours and clinical/laboratory assessments. Multiple regression analyses were performed to identify determinants of physical and mental HRQoL in the overall population and their potential differential effects by ethnicity. No a priori hypotheses were formulated so all interaction effects were explored.

Principal Findings

HRQoL levels differed between ethnic groups. Chinese respondents had higher physical HRQoL (PCS) than Indian and Malay participants (p<0.001) whereas mental HRQoL (MCS) was higher in Malay relative to Chinese participants (p<0.001). Regressions models explained 17.1% and 14.6% of variance in PCS and MCS respectively with comorbid burden, income and employment being associated with lower HRQoL. Age and family were associated only with MCS. The effects of gender, stroke and musculoskeletal conditions on PCS varied by ethnicity, suggesting non-uniform patterns of association for Chinese, Malay and Indian individuals.

Conclusions

Differences in HRQoL levels and determinants of HRQoL among ethnic groups underscore the need to better or differentially target population segments to promote well-being. More work is needed to explore HRQoL and wellness in relation to ethnicity.     

Probiotic Activity of a Bacterial Strain Isolated from Ancient Permafrost Against Salmonella Infection in Mice

Bacillus cereus strain F, collected from relict permafrost located in Siberia, was analyzed for probiotic activity in the mouse Salmonella enterica model. Viable bacterial cells were found in frozen soils taken at Mammoth Mountain in Yakutia from a depth below the level of seasonal thawing. Geological data indicated the absence of a thawing within millions of years of deposited soils, which helped to ensure the ancient origin of our sample. According to DNA analysis, bacterial cells collected from the relict permafrost appeared to be B. cereus strain F. The morphology of these bacteria was analyzed using atomic force microscopy. B. cereus strain F was assessed as a nonpathogenic bacterium by evaluation of its pathogenicity. A S. enterica model is described in mice after per oral inoculation and serves as a model for the human carrier state. Using this model, probiotic activity by the bacterial strain isolated from the ancient permafrost has been shown against Salmonella infection in mice.     

Rates of Intentional and Unintentional Nonadherence to Peritoneal Dialysis Regimes and Associated Factors

With increasing emphasis on expanding home-based dialysis, there is a need to understand adherence outcomes. This study set out to examine the prevalence and predictors of nonadherence among patients undergoing peritoneal dialysis. A cross sectional sample of 201 peritoneal dialysis patients recruited between 2010–2011 from Singapore General Hospital completed measures of quality of life, medication beliefs, self-efficacy and emotional distress. Nonadherence rates were high; 18% for dialysis, 46% for medication and 78% for diet. Intentional nonadherence was more common for dialysis (p = .03), whereas unintentional nonadherence was more common for medication (p = .002). Multivariate models indicated significant associations for higher education (intermediate vs low OR = 3.18, high vs low OR = 4.70), lower environment quality of life (OR = 0.79), dialysis self-efficacy (OR = 0.80) with dialysis nonadherence; higher education (OR = 2.22), self-care peritoneal dialysis (OR = 3.10), perceived necessity vs concerns over medication (OR = 0.90), self-efficacy (OR = 0.76) with nonadherence to medication. The odds for nonadherence to diet were higher among patients who were younger (OR = 0.96), of Chinese ethnicity (OR = 2.99) and those reporting better physical health (OR = 1.30) and lower self-efficacy (OR = 0.49). Nonadherence is common in peritoneal dialysis. Self-efficacy and beliefs about medication are promising targets for interventions designed to improve adherence.     

Phenol hydroxylase from Acinetobacter radioresistens S13. Isolation and characterization of the regulatory component.

This paper reports the isolation and characterization of the regulatory moiety of the multicomponent enzyme phenol hydroxylase from Acinetobacter radioresistens S13 grown on phenol as the only carbon and energy source. The whole enzyme comprises an oxygenase moiety (PHO), a reductase moiety (PHR) and a regulatory moiety (PHI). PHR contains one FAD and one iron-sulfur cluster, whose function is electron transfer from NADH to the dinuclear iron centre of the oxygenase. PHI is required for catalysis of the conversion of phenol to catechol in vitro, but is not required for PHR activity towards alternative electron acceptors such as cytochrome c and Nitro Blue Tetrazolium. The molecular mass of PHI was determined to be 10 kDa by SDS/PAGE, 8.8 kDa by MALDI-TOF spectrometry and 18 kDa by gel-permeation. This finding suggests that the protein in its native state is a homodimer. The isoelectric point is 4.1. PHI does not contain any redox cofactor and does not bind ANS, a fluorescent probe for hydrophobic sites. The N-terminal sequence is similar to those of the regulatory proteins of phenol hydroxylase from A. calcoaceticus and Pseudomonas CF 600. In the reconstituted system, optimal reaction rate was achieved when the stoichiometry of the components was 2 PHR monomers: 1 PHI dimer: 1 PHO (alphabetagamma) dimer. PHI interacts specifically with PHR, promoting the enhancement of FAD fluorescence emission. This signal is diagnostic of a conformational change of PHR that might result in a better alignment with respect to PHO.