Potential biomarkers of ageing

Life span in individual humans is very heterogeneous.Thus, the ageing rate, measured as the decline of functional capacity and stress resistance, is different in every individual. There have been attempts made to analyse this individual age, the so-called biological age, in comparison to chronological age. Biomarkers of ageing should help to characterise this biological age and, as age is a major risk factor in many degenerative diseases,could be subsequently used to identify individuals at high risk of developing age-associated diseases or disabilities.Markers based on oxidative stress, protein glycation,inflammation, cellular senescence and hormonal deregulation are discussed.     

Sensitive determination of ciprofloxacin and norfloxacin in biological fluids using an enzymatic rotating biosensor

The high sensitivity that can be attained using an enzymatic system and mediated by catechol has been verified by on-line interfacing of a rotating biosensor and continuous-flow/stopped-flow/continuous-flow processing. Horseradish peroxidase, HRP, [EC 1.11.1.7], immobilized on a rotating disk, in the presence of hydrogen peroxide catalyzed the oxidation of catechol, whose back electrochemical reduction was detected on glassy carbon electrode surface at -200mV. Thus, when ciprofloxacin (CF) or norfloxacin (NF) was added to the solution, these piperazine-containing compounds participate in Michael addition reactions with catechol to form the corresponding aminoquinone derivative, decreasing the peak current obtained in proportion with the increase of its concentration. CF was used as the model piperazine-containing compound for the study. The influence of indicator composition on the nature of the analytical response has been assessed through examining the electrochemical properties of three derivatives. Interference by electroactive species (ascorbate, urate, and tyrosine) and other physiological constituents (cysteine, glutathione) has also been assessed.     

GALNT11 as a new molecular marker in chronic lymphocytic leukemia

Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B–CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [?²(1) = 18.26; P < 0.0001], lipoprotein lipase expression [?²(1) = 13.72; P = 0.0002] and disease prognosis [?²(1) = 15.49; P < 0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Interrelationship between phosphorus toxicity and sugar metabolism in Verticordia plumosa L

Phosphorus, an essential plant nutrient, may become toxic when accumulated by plants to high concentrations. Certain plant species such as Verticordia plumosa L. suffer from P toxicity at solution concentrations far lower than most other plant species. In this study, exposure of V. plumosa plants to a solution containing as low as 3 mg l^–1 P resulted in significant growth inhibition and typical symptoms of P toxicity. In a wide range of P levels studied, micronutrient concentrations in V. plumosa leaves were within the range considered adequate for optimal growth. Notably, tomato plants with high hexokinase activity due to overexpression of Arabidopsis hexokinase (AtHXK1) exhibited senescence symptoms similar to those of P toxic V. plumosa. The resemblance in senescence symptoms between P-toxic tomato plants and those with high hexokinase activity suggested that increased sugar metabolism could play a role in P toxicity in plants. To test this hypothesis, we determined the amount of hexose phosphate, the product of hexokinase, in V. plumosa leaves grown at various P levels in the nutrient solution. Positive correlations were found between concentration in the medium, P concentration in the plant, hexose phosphate concentration in leaves and P toxicity symptoms. Foliar Zn application suppressed P toxicity symptoms and reduced the level of hexose phosphate in leaves. Furthermore, Zn also inhibited hexokinase activity in vitro. Based on these results we suggest that P toxicity involves sugar metabolism via increased activity of hexokinase that accelerates senescence     

Adaptation of a gamma-glutamyl transpeptidase assay to microtiter plates

A kinetic assay for measuring gamma-glutamyl transpeptidase (GGT) activity has been adapted to microtiter plates and an automated microtiter plate reader. This method permits the simultaneous analysis of enzyme activity in a large number of samples incubated with the chromogenic GGT substrate gamma-glutamyl-p-nitroanilide. A major advantage of this assay over previously reported methods is the substantial reduction in the time needed for measuring sample enzyme activity. In addition, reduction of the total assay volume to 0.28 ml conserves both sample and reagents. This method has been calibrated at 23 degrees C using purified GGT, and used to analyze GGT activity in human sera. The assay is sensitive over a range of 3-200 U/liter.     

Dihydrofolate reductase activity and resistance to aminopterin in various species of Drosophila

Summary The aim of our work was to compare the mechanisms of resistance to aminopterin, inhibitor of the dihydrofolate reductase enzyme, between different Drosophila species and those described for cultured cells. Moreover we compared the systematic species divisions based on morphological traits and those based on a molecular approach. For this purpose, the effect of aminopterin on viability and wing phenotype was studied in different Drosophila species. Dihydrofolate reductase was measured in adult flies. We found an important dihydrofolate reductase activity in the melanogaster sub-group compared to the other species studies. Wing effect was observed only in this sub-group. The effects of aminopterin on the wing phenotype were very similar to the phenotype of rudimentary mutants. Both deplete the pyrimidine pool and it has been shown by the studies of the structural genes of the nucleotide pyrimidine pathway that the wing tissue is very sensitive to every pertubation of this metabolism.The D. ananassae species was found to be fully resistant at the concentrations of the inhibitor tested. No or very little dihydrofolate reductase activity was detected. The binding of the enzyme to the inhibitor was comparable to that found in the Oregon strain of D. melanogaster. The purine and pyrimidine salvage pathways were investigated and the D. ananassae species displayed an important thymidine kinase activity. The D. ananassae flies were sensitive on Sang medium compared to the Oregon flies but were able to use exogenous bases or nucleosides more efficiently. Therefore the mechanism of resistance to aminopterin in Drosophila may be different from those described for methotrexate in mammalian cultured cells, as indicated by the results obtained for D. ananassae.