Two different clean-up procedures for liquid chromatographic determination of ochratoxin A in urine

This paper describes two different procedures for extraction of ochratoxin A (OTA) from urine samples: one using acidic chloroform-methanol mixture, followed by solid-phase extraction (SPE) clean-up and the other using commercial Chem Elut columns and a chloroform-formic acid mixture. The recovery of OTA using the procedure with silica gel columns was 82% with a R.S.D. < 8.4% and the detection and quantitation limits were 0.5 and 1.5 ng OTA/ml, respectively. The recovery of OTA in the second procedure with urine samples purified only on commercial Chem Elut columns was 95% with R.S.D. < 4.0%, and detection and quantitation limits 0.3 and 0.9 ng/ml, respectively. Both procedures of OTA extraction effectively eliminate interfering substances and give reliable and repeatable results. However, the procedure with Chem Elut columns gave higher recovery and lower detection and quantitation limits. It was successfully applied in determining OTA in human urine samples.     

Celiac Disease–Specific TG2-Targeted Autoantibodies Inhibit Angiogenesis Ex Vivo and In Vivo in Mice by Interfering with Endothelial Cell Dynamics

A characteristic feature of celiac disease is the presence of circulating autoantibodies targeted against transglutaminase 2 (TG2), reputed to have a function in angiogenesis. In this study we investigated whether TG2-specific autoantibodies derived from celiac patients inhibit angiogenesis in both ex vivo and in vivo models and sought to clarify the mechanism behind this phenomenon. We used the ex vivo murine aorta-ring and the in vivo mouse matrigel-plug assays to address aforementioned issues. We found angiogenesis to be impaired as a result of celiac disease antibody supplementation in both systems. Our results also showed the dynamics of endothelial cells was affected in the presence of celiac antibodies. In the in vivo angiogenesis assays, the vessels formed were able to transport blood despite impairment of functionality after treatment with celiac autoantibodies, as revealed by positron emission tomography. We conclude that celiac autoantibodies inhibit angiogenesis ex vivo and in vivo and impair vascular functionality. Our data suggest that the anti-angiogenic mechanism of the celiac disease-specific autoantibodies involves extracellular TG2 and inhibited endothelial cell mobility.     

Fumonisin B1 inhibits mitochondrial respiration and deregulates calcium homeostasis--implication to mechanism of cell toxicity

Fumonisin B(1) (FB(1)) is a neurodegenerative mycotoxin produced by Fusarium verticiloides mould that contaminates maize worldwide. FB(1) toxicity has been connected with deregulation of sphingolipid metabolism, but the mechanism of cytotoxicity remains controversial. In cell cultures of rat primary astrocytes and human neuroblastoma (SH-SY5Y), we found that FB(1) inhibits mitochondrial complex I, which leads to a decrease in the rate of mitochondrial and cellular respiration, depolarisation of the mitochondrial membrane, induction of reactive oxygen species (ROS) production in mitochondria and deregulation of calcium signalling. Despite the increase in ROS production, the intracellular level of glutathione (GSH) was significantly increased. After 24h of FB(1) exposure, no cell death was observed. Thus, mitochondria appear to be the primary target of FB(1), which leads to sustained deregulation of calcium homeostasis and presumably to cell death.     

Developmental and transplacental genotoxicology: fluconazole

Over the last 40 years mankind has been facing new types of radiochemical environmental settings with every decade. During the last decade, biomonitoring was additionally focused on assessing associations between environmental exposure(s) and both early and late biological effects in children. Despite efforts to control and avoid child exposure to genotoxic agents the incidence of childhood cancers is increasing. Some cancers in adulthood may be the consequence of a multi-step process which starts with intrauterine and childhood exposure. This highlights the importance of a comprehensive interpretation of multiple health effects, especially considering recent studies suggesting that most health disorders are related to DNA changes. When exposed to genotoxic agents, a developing organism (fetus or child) is constantly being forced to reorganize into new equilibriums in order to adjust to a xenobiotic environment. In addition, the influence of sex hormones on radiochemical sensitivity is still unknown. For this reason special attention should be paid to puberty. The results of recent studies on animal models and follow up studies on children after nuclear accidents show long-lasting cytogenetic damage even after low dose exposures and their transgenerational persistance. To evaluate age-related difference and transplacental genotoxic potency fluconazole (FC) was investigated by in vivo micronucleus (MN) assay in adult mice, young mice and in transplacentally exposed newborn pups. Compared to the baseline values, FC caused no detectable genome damage in adult animals, but there was a significant increase in MN frequency in young animals and in newborn pups. Our study thus exemplifies an age-related chemosensitivity, and argues that cancer-promoting disturbances of complex prenatal developmental mechanisms and maturation during childhood require a new approach using systems biology.     

Systemic redox imbalance in stable chronic obstructive pulmonary disease

Context: Increased oxidative burden is found in chronic obstructive pulmonary disease (COPD).

Objective: To assess the association of ceruloplasmin, albumin, bilirubin, transferrin, thiols and malondialdehyde (MDA) with stable COPD.

Materials and methods: Oxidative stress markers measured in 106 COPD patients and 45 healthy subjects were evaluated.

Results: Higher ceruloplasmin and MDA, and lower albumin, transferrin and thiols in COPD patients were found. Ceruloplasmin was the strongest single predictor of COPD. The model combining ceruloplasmin, albumin and thiols improved their individual diagnostic performances.

Conclusions: Diagnostic characteristics of ceruloplasmin, albumin, transferrin, thiols and MDA suggest their potential value as additional tools in disease diagnosis.     

Rapid interactome profiling by massive sequencing

We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120 000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.