Serum paraoxonase-1 (PON1) activities (PONase/AREase) and polymorphisms in patients with type 2 diabetes mellitus in a North-West Indian population

Objective

Paraoxonase-1 (PON1), an HDL-C associated enzyme, protects lipoproteins from oxidation. There is evidence that PON1 enzyme activity is reduced in the patients with type 2 diabetes mellitus (T2DM). North-West Indian Punjabis, a distinct ethnic group has high incidence of T2DM. However till date there is no information regarding PON1 enzyme activities and PON1 polymorphisms in T2DM patients of this ethnic group.

Methods

We identified polymorphisms in the coding Q192R, L55M and promoter − 909G/C, − 162A/G, − 108C/T of the PON1 gene by using PCR-RFLP, multiplex PCR and allele specific oligonucleotide PCR assays in 250 T2DM patients and 300 healthy controls. We also assessed paraoxonase (PONase) and arylesterase (AREase) activities of PON1 enzyme.

Results

The serum PONase (114.2 vs. 178.0 nmol/min/ml) and AREase (62.7 vs. 82.5 μmol/min/ml) activities were significantly lower (p < 0.0001) in patients as compared to controls. PONase activity was affected by all the studied PON1 polymorphisms. However, AREase activity was not affected by any of these polymorphisms. Coding Q192R and promoter − 909G/C polymorphisms showed significant differences in genotypic distribution. QR, RR (Q192R) and GC, CC (− 909G/C) genotypes and L-C-A-R-G, L-T-A-R-G, L-T-G-Q-C haplotypes showed significant association with type 2 diabetes. No significant linkage disequilibrium was observed among the five polymorphisms.

Conclusion

Both PONase and AREase activities are lower in patients and this could lead to increased lipid peroxidation and accelerated atherosclerosis in them. PONase activity, but not AREase activity is influenced by PON1 polymorphisms. QR, RR, GC, CC genotypes and L-C-A-R-G, L-T-A-R-G, L-T-G-Q-C haplotypes are commoner in diabetics as compared to controls and may be related to genetic susceptibility to type 2 diabetes.     

The diagnostic accuracy of high-mobility group box 1 protein and twelve other markers in discriminating bacterial, viral and co-infected bronchial pneumonia in Han children

Pneumonia in children is common and can lead to grave consequences if not addressed in a proper and timely manner. In the management of pneumonia, early identification of the causative infective agent is of obvious importance for treatment, as it allows selection of the appropriate antibiotics. However, such identification requires laboratory test results, which may not be immediately available. The aim of this study was to evaluate the accuracy and usefulness of 13 markers in differentiating between viral and bacterial pneumonia in Han children (34 healthy controls and 78 patients). It was found that WBC counts were more accurate in diagnosis of the type of agent responsible for infection than was the degree of expression of HMGB1. Among the 13 markers investigated, HMGB1 was the best at discriminating between co-infected (bacterium and virus) and single-infected (bacterium or virus) children with bronchial pneumonia. HMGB1 expression of less than 1.0256, excluded most co-infections (the negative predictive value was greater than 89.7%). Diagnosed sole viral pneumonia clinically overlapped with bacterial pneumonia, but bacterial pneumonia was more often associated with higher white blood cell (WBC) counts (WBC ≥ 13,000 cells/mm(3)). When the two marker readouts--HMGB1 < 1.0256 and WBC ≥ 13,000 cells/mm(3)--were combined, the positive predictive value for bacterial pneumonia alone was 92.3%. These findings can help clinicians discriminate between bronchial pneumonia caused by virus, bacterium or both with a high specificity.     

High glucose concentration affects the oxidant-antioxidant balance in cultured mouse podocytes

Hyperglycemia is well-recognized and has long-term complications in diabetes mellitus and diabetic nephropathy. In podocytes, the main component of the glomerular barrier, overproduction of reactive oxygen species (ROS) in the presence of high glucose induces dysfunction and increases excretion of albumin in urine. This suggests an impaired antioxidant defense system has a role in the pathogenesis of diabetic nephropathy. We studied expression of NAD(P)H oxidase subunits by Western blotting and immunofluorescence and the activities of the oxidant enzyme, NAD(P)H, and antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), in mouse podocytes cultured in a high glucose concentration (30 mM). We found long-term (3 and 5 days) exposure of mouse podocytes to high glucose concentrations caused oxidative stress, as evidenced by increased expression of Nox4 and activities of NAD(P)H oxidase (Δ 182%) and SOD (Δ 39%) and decreased activities of GPx (Δ -40%) and CAT (Δ -35%). These biochemical changes were accompanied by a rise in intracellular ROS production and accumulation of hydrogen peroxide in extracellular space. The role of Nox4 in ROS generation was confirmed with Nox4 siRNA. In conclusion, high glucose concentration affects the oxidant-antioxidant balance in mouse podocytes, resulting in enhanced generation of superoxide anions and its attenuated metabolism. These observations suggest free radicals may play an important role in the pathogenesis of diabetic nephropathy.     

The application of atmospheric pressure matrix-assisted laser desorption/ionization to the analysis of long-term cryopreserved serum peptidome

Although most time-of-flight (TOF) mass spectrometers come equipped with vacuum matrix-assisted laser desorption/ionization (MALDI) sources, the atmospheric pressure MALDI (API–MALDI) source is an attractive option because of its ability to be coupled to a wide range of analyzers. This article describes the use of an API–MALDI source coupled to a TOF mass spectrometer for evaluation of the effects of medium- and long-term storage on peptidomic profiles of cryopreserved serum samples from healthy women. Peptides were purified using superparamagnetic beads either from fresh sera or after serum storage at −80 °C for 18 months or at −20 °C for 8 years. Data were preprocessed using newly developed bioinformatic tools and then were subjected to statistical analysis and class prediction. The analyses showed a dramatic effect of storage on the abundance of several peptides such as fibrinopeptides A and B, complement fractions, bradykinin, and clusterin, indicated by other authors as disease biomarkers. Most of these results were confirmed by shadow clustering analysis, able to classify each sample in the correct group. In addition to demonstrating the suitability of the API–MALDI technique for peptidome profiling studies, our data are of relevance for retrospective studies that involve frozen sera stored for many years in biobanks.     

Elucidation of the mechanism of N-demethylation catalyzed by cytochrome P450 monooxygenase is facilitated by exploiting nitrogen-15 heavy isotope effects

Tryptophan indole-lyase (Trpase), PBPRA2532, from Photobacterium profundum SS9, a piezophilic marine bacterium, has been cloned, expressed in Escherichia coli, and purified. The P. profundum Trpase (PpTrpase) exhibits similar substrate specificity as the enzyme from E. coli (EcTrpase). PpTrpase has an optimum temperature for activity at about 30 °C, compared with 53 °C for EcTrpase, and loses activity rapidly (t_1/2 ∼ 30 min) when incubated at 50 °C, while EcTrpase is stable up to 65 °C. PpTrpase retains complete activity when incubated more than 3 h at 0 °C, while EcTrpase has only about 20% remaining activity. Under hydrostatic pressure, PpTrpase remains fully active up to 100 MPa (986 atm), while EcTrpase exhibits only about 10% activity at 100 MPa. PpTrpase forms external aldimine and quinonoid intermediates in stopped-flow experiments with l-Trp, S-Et-l-Cys, S-benzyl-l-Cys, oxindolyl-l-Ala, l-Ala and l-Met, similar to EcTrpase. However, with l-Trp a gem-diamine is observed that decays to a quinonoid complex. An aminoacrylate is observed with l-Trp in the presence of benzimidazole, as was seen previously with EcTrpase [28] but not with S-Et-l-Cys. The results show that PpTrpase is adapted for optimal activity in the low temperature, high pressure marine environment.     

Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

In Synechocystis sp. PCC 6803, the loop domain (aa 1–70) of the phycobilisome core-membrane linker, L_CM, was found to interact with the glycosyl transferase homolog, Sll1466. Growth of a Sll1466 knock-out mutant was slightly faster in low light, but strongly inhibited in high light; the phenotype is discussed in relation to the regulation of light energy transfer to photosystem II. At the molecular level, the mutant shows the following changes compared to the wild type: (1) a smaller size and higher mobility of phycobilisomes on the thylakoid membrane, and (2) a changed lipid composition of the thylakoid membrane, especially decreased amounts of digalactosyl diacylglycerol. These results indicate a profound regulatory role for Sll1466 in regulating photosynthetic energy transfer.     

Root-fed salicylic acid in grape involves the response caused by aboveground high temperature

In order to investigate the transportation and distribution of salicylic acid (SA) from root to aboveground tissues in response to high temperature, the roots of grape plant were fed with 14C-SA before high temperature treatment. Radioactivity results showed that progressive increase in SA transportation from root to aboveground as compared with the control varied exactly with the heat treatment time. Radioactivity results of leaves at different stem heights indicated that the increase in SA amount at the top and middle leaves during the early period was most significant in comparison with the bottom leaves. The up-transportation of SA from root to aboveground tissues was dependent on xylem rather than phloem. Auto-radiographs of whole grape plants strongly approved the conclusions drawn above. Root-derived SA was believed to be a fundamental source in response to aboveground high temperature.     

An objective, rapid and reproducible method for scoring AFLP peak-height data that minimizes genotyping error

Amplified fragment length polymorphism (AFLP) fingerprint data are now commonly collected using DNA sequencers. AFLP genotypes are still often scored by eye from such data - a time-consuming, error-prone and subjective process. We present a semi-automated method of genotyping sequencer-collected AFLPs at predefined fragment locations (loci) within the fingerprint. Our method uses thresholds of AFLP-polymerase chain reaction-product fluorescence intensity (peak height) in order to: (i) exclude AFLP loci that are likely to contribute high rates of error to data sets, and (ii) determine the AFLP phenotype (fragment presence or absence) at the retained loci. Error rate analysis is an integral part of this process and is used to determine optimal thresholds that minimize genotyping error, while maximizing the numbers of retained loci. We show that application of this method to a large AFLP data set allows genotype calls that are rapid, objective and repeatable, facilitating the extraction of reliable genotype data for molecular ecological studies.