Paraoxonase 1 status in keratoconus: a preliminary study of activity and polymorphism / Estatus de la paraoxonasa 1 en el queratocono: estudio preliminar de actividad y polimorfismo

OBJECTIVE: To determine the activity of paraoxonase 1 (PON1) in keratoconus in a Malaysian population in comparison with non-keratoconic subjects. METHODS: Clinical eye examinations were performed on patients with keratoconus and non-keratoconic subjects after questionnaires were completed. Blood samples were collected and subjected to spectrophotometry analysis of paraoxonase and diazoxonase activities for the determination of the status of PON1 of every individual. RESULTS: Of the 11 keratoconic patients and 55 non-keratoconic control samples collected, eight patients of Indian ethnicity were keratoconic (73%), whereas 33 non-Indians were non-keratoconic (60%; p = 0.047). Paraoxonase activity was lower in Indians compared to the non-Indians ie Malays and Chinese (p = 0.008). Keratoconic subjects had a lower paraoxonase activity compared to non-keratoconics (p = 0.038). CONCLUSIONS: The reduced paraoxonase activity in keratoconic patients suggests that the keratoconic corneas were more susceptible to oxidative stress. Reduced paraoxonase activity and keratoconus status appears to be associated with ethnicity.

OBJETIVO: Determinar la actividad de paraoxonasa 1 (Pon 1) en el queratocono en una población malaya, en comparación con sujetos no queratocónicos. MÉTODOS: Se realizaron exámenes clínicos oculares a pacientes con queratocono y a sujetos no queratocónicos luego que los mismos respondieran a los cuestionarios. Se recogieron muestras de sangre, que fueron entonces sometidas a análisis espectrofotométrico en relación con las actividades de la paraoxonasa y la diazoxonasa para la determinación del estatus de la paraoxonasa 1 de cada individuo. RESULTADOS: De los 11 pacientes queratocónicos y las 55 muestras de control no queratocónicas recogidas, 8 pacientes de etnicidad india fueron queratocónicos (73%), mientras que 33 no indios fueron no queratocónicos (60%; p = 0.047). La actividad de la paraoxonasa fue más baja en los indios en comparación con los no indios, es decir, los malayos y los chinos (p = 0.008). Los sujetos queratocónicos tenían una actividad de la paraoxonasa más baja, comparada con los no queratocónicos (p = 0.038). CONCLUSIONES: La actividad de la paraoxonasa reducida en los pacientes queratocónicos sugiere que las córneas queratocónicas son más susceptibles al estrés oxidativo. La actividad de la paraoxonasa reducida y el estatus del queratocono parecen estar asociados con la etnicidad.

Lack of association between Gly82Ser, 1704G/T and 2184A/G of RAGE gene polymorphisms and retinopathy susceptibility in Malaysian diabetic patients.

Diabetic retinopathy is the most common diabetic eye disease, occurring in about 60% of type 2 diabetic patients. Other than known clinical risk factors, the influence of genes has been suggested as part of the development of diabetic retinopathy. We investigated the association of Gly82Ser, 1704G/T and 2184A/G polymorphisms in the RAGE gene with retinopathy in type 2 diabetic patients in Malaysia. Ninety-eight unrelated retinopathy patients and 185 unrelated healthy controls from all over Malaysia were recruited in this study. The allele and genotype frequencies of the three gene polymorphisms were investigated using PCR-RFLP. The allele frequency of the three polymorphisms did not differ significantly between the control and the retinopathy group (P > 0.05). Analysis of the frequency of GA+AA, GT+TT and AG+GG in the retinopathy group did not reveal significant differences (P > 0.05) compared to the control group. We conclude that RAGE gene Gly82Ser, 1704G/T and 2184A/G polymorphisms are not associated with retinopathy development in the Malaysian population.

Paraoxonase 1 status in keratoconus: a preliminary study of activity and polymorphism.

OBJECTIVE: To determine the activity of paraoxonase 1 (PON1) in keratoconus in a Malaysian population in comparison with non-keratoconic subjects. METHODS: Clinical eye examinations were performed on patients with keratoconus and non-keratoconic subjects after questionnaires were completed. Blood samples were collected and subjected to spectrophotometric analysis of paraoxonase and diazoxonase activities for the determination of the status of PON1 of every individual. RESULTS: Of the 11 keratoconic patients and 55 non-keratoconic control samples collected, eight patients of Indian ethnicity were keratoconic (73%), whereas 33 non-Indians were non-keratoconic (60%; p = 0.047). Paraoxonase activity was lower in Indians compared to the non-Indians ie Malays and Chinese (p = 0.008). Keratoconic subjects had a lower paraoxonase activity compared to non-keratoconics (p = 0.038). CONCLUSIONS: The reduced paraoxonase activity in keratoconic patients suggests that the keratoconic corneas were more susceptible to oxidative stress. Reduced paraoxonase activity and keratoconus status appears to be associated with ethnicity.

A mouse model of lamellar intrastromal femtosecond laser keratotomy: ultra-structural, inflammatory, and wound healing responses.

PURPOSE: The availability of knockout mouse species provide a highly versatile platform for critically examining the corneal wound healing response. We aimed to develop and characterize the wound healing response in a mouse model of intrastromal femtosecond laser (FSL) keratotomy. METHODS: An intrastromal lamellar dissection using a Visumax FSL was performed on 16 wild type mice (C57BL6) . The energy level was optimized at 150nJ. The FSL was programmed to perform a lamellar dissection at 50 µM depth without sidecut. The flap was not lifted. Fellow eyes were used as controls. Slit lamp photography and confocal microscopy were performed immediately before the mice were sacrificed 4 h, 1, 3, and 7 days post surgery. Corneas were harvested for immunocytochemistry, transmission electron microscopy (TEM) and light microscopy (LM). RESULTS: Confocal microscopy showed an absence of keratocytes in the area immediately surrounding the dissection plane. The dissection plane and individual FSL plasma cavitation bubbles were clearly evident on TEM. There was evidence of Keratocyte cell death along the laser resection plane on TEM. LM revealed the dissection plane at a 20 µM depth, although not all epithelial cell layers were intact. Staining for monocytes using antibodies for CD11b (cluster of differentiation 11b) showed early migration at the peripheries at 4 h that increased at 24 h and became more central in treated corneas (p<0.001). Apoptotic cells were evident on TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay in the immediate ablation zone and were significantly raised at 4 and 24 h (p<0.001). Ki67 (Kiel 67 protein) positive proliferating keratocytes are evident at 3 days and increased significantly by 7 days (p<0.001). Minimal fibroblast (cluster of differentiation 90, CD90) transformation was seen at 1 week. No myofibroblasts were detected. DISCUSSION: We have demonstrated that FSL lamellar cuts can be effectively performed on mice and that this model exhibits typical signs of the corneal wound healing response. This model could provide a ubiquitous platform in which to study corneal wound healing responses in both wild type and knockout mice species. The ability to create such a lamellar pocket may be utilizzd for intrastromal drug delivery.

An in vitro comparison of the bleaching efficacy of 35% carbamide peroxide with established intracoronal bleaching agents.

AIM: To evaluate the bleaching efficacy of 35% carbamide peroxide, 35% hydrogen peroxide and sodium perborate for intracoronal bleaching of root filled discoloured teeth. METHODOLOGY: Extracted premolars were artificially stained using whole blood then root canal treatment was performed. After obturation, a 2 mm intermediate base was placed 1 mm below the buccal amelo-cemental junction. Intracoronal bleaching was performed in 11 teeth per group, using either 35% carbamide peroxide gel (group CP), 35% hydrogen peroxide gel (group HP) or sodium perborate mixed with distilled water (group SP). The bleaching agents were replaced after 7 days. The shade of the teeth was evaluated at day 0, 7 and 14. The results were analysed using Kruskal-Wallis one-way analysis of variance and Mann-Whitney U-test. RESULTS: At the end of 7 days, both groups CP and HP lightened by 8 +/- 3 Vita tab positions, respectively, whereas group SP lightened by 5 +/- 3 tab positions (P < 0.05). At the end of the second bleaching period at day 14, group CP and HP lightened by a further 2 +/- 2 and 2 +/- 3 tab positions, respectively, whereas group SP lightened by a further 3 +/- 4 tab positions. There were no statistical differences between groups at day 14. CONCLUSIONS: Thirty-five per cent carbamide peroxide and 35% hydrogen peroxide were equally effective for intracoronal bleaching, and significantly better than sodium perborate after 7 days. After 14 days, there were no significant differences between the groups. Thirty-five per cent carbamide peroxide can be recommended as an equally effective alternative to hydrogen peroxide for intracoronal bleaching.

Extraradicular diffusion of hydrogen peroxide and pH changes associated with intracoronal bleaching of discoloured teeth using different bleaching agents.

AIM: To evaluate the extraradicular pH and hydrogen peroxide (HP) diffusion when either 35% carbamide peroxide (CP), 35% HP or sodium perborate (SP) is used for intracoronal bleaching of artificially discoloured teeth. METHODOLOGY: Single rooted extracted human premolars were stained with whole blood cells. After shaping and cleaning, they were root filled and a base cement placed 1 mm below the buccal cementoenamel junction (CEJ). Four cemental defects were prepared just below the CEJ on each root surface. The teeth were randomly divided into four groups of 11 specimens, and intracoronally bleached using CP, HP, SP or distilled water (CL). Each tooth was suspended in a vial of distilled water and bleached for 7 days. The pH of the extraradicular distilled water was tested at 0, 1, 2 and 7 days and the HP that diffused through the root quantified using the Ferrous Oxidation-Xylenol Orange 2 Assay. The results were analysed using the one-way anova and Scheffe tests. RESULTS: Carbamide peroxide produced the greatest increase and HP the least pH change (P < 0.05 except day 1), SP was intermediate. From day 1 onwards, radicular diffusion of HP was greatest with HP and least with CP (P < 0.01), again SP was intermediate. There was no significant difference between CP and SP. CONCLUSIONS: Carbamide peroxide had very low levels of extraradicular diffusion of HP, in the presence of cemental defects. It could be an alternative to the other intracoronal bleaching agents.

Complete characterisation of Tn5530 from Burkholderia cepacia strain 2a (pIJB1) and studies of 2,4-dichlorophenoxyacetate uptake by the organism.

The complete genetic characterisation of Tn5530 in Burkholderia cepacia strain 2a (pIJB1) has been accomplished, indicating that it is a Tn3-like transposon with a complex structure bearing operons for the catabolism of 2,4-dichlorophenoxyacetate (2,4-D) and malonate. Tn5530 is terminated at both ends by the IS1071::IS1471 element and the 2,4-D- and malonate-dissimilatory operons are separated by a region encoding a putA and lrp gene and a gene encoding a chloride channel protein. The chloride channel protein may have a role in the expulsion of chloride ions liberated by the dissimilation of 2,4-D. In addition, a putative transposase with a high level of sequence similarity to those of plasmid pGH1 from Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. glycinea, and a transcription factor similar to those of the TetR family with low but significant levels of sequence similarity to those identified in a number of other organisms was observed. The entire Tn5530 sequence length, including the IS1071::IS1471 elements, was found to be 40,956bp, and pIJB1 was replicon-typed and otherwise characterised as being of the IncP-1beta subgroup, bearing merA and merD genes conferring resistance to mercuric chloride. The rate of uptake of 2,4-D by B. cepacia strain 2a was observed to proceed more readily at acid pH, suggesting involvement of the undissociated form of the compound. Uptake did not show saturation kinetics, was concentration-dependent, and appeared to occur in two stages; an initial accumulation followed by a linear second phase. Uptake could be inhibited by sodium azide but not by arsenate, N,N(')-dicyclohexylcarbodi-imide (DCCD) or carbonylcyanide m-chlorophenyl-hydrazone (CCCP) suggesting that it is not energy-dependent.

2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenases from Burkholderia cepacia 2a and Ralstonia eutropha JMP134.

2,4-Dichlorophenoxyacetate (2,4-D)/alpha-ketoglutarate (alpha-KG) dioxygenase has been purified to apparent homogeneity from Burkholderia cepacia strain 2a, which utilizes 2,4-D as sole carbon source. The enzyme required ferrous ions, and was a homodimer composed of subunits having an Mr of approximately 32,000. The reaction catalysed consumed one mol each of 2,4-D, alpha-KG and dioxygen, with the production of one mol each of succinate, 2,4-dichlorophenol and glyoxylate. Maximum activity was exhibited at pH 7.8 and 25 degrees C, and reactivity was enhanced by the presence of ascorbate and cysteine. Mn2+, Zn2+, Cu2+, Fe3+ and Co2+ were inhibitory, and chemical modification of the dioxygenase revealed that thiol groups were essential for activity. The enzyme was active towards other substituted phenoxyacetates, but reacted most rapidly with 2,4-D. The apparent Michaelis constants for 2,4-D and alpha-KG were 109 and 8.9 microM, respectively. The properties of this enzyme are compared with those of the 2,4-D/alpha-KG dioxygenase from Ralstonia eutropha JMP134, which exhibits a differing N-terminal amino-acid sequence, and a different temperature 'optimum', pH optimum, substrate specificity and sensitivity to thiol-binding reagents.