Discovery of large molecules as new biomarkers in wastewater using environmental proteomics and suitable polymer probes.

The capability of monitoring large molecules as possible biomarkers in wastewater will be an important contribution to the new field of sewage epidemiology. Here, we explore the use of polymer probes together with untargeted proteomics for large scale protein analysis in sewage and treated water. Polymeric probes were immersed in the influent, anoxic reactor and effluent waters of a Spanish WWTP during 11 days. Proteins sorbed were extracted and identified by mass spectrometry. A total of 690 proteins from bacteria, plants and animals, including human, were identified showing different proteome profiles in the different sites. Bacterial proteins (510) pointed at 175 genera distributed in 22 bacterial classes. The most abundant were EF-Tu, GroEL and ATP synthase which were contributed by a high number of species. Human was the species contributing the greatest number of identified proteins (57), some in high abundance like keratins. Human proteins dominated in the influent water and were efficiently removed at the effluent. Several of the proteins identified (S100A8, uromodulin, defensins) are known disease biomarkers. This study provides the first insight into the proteome profiles present in real wastewater.

Acute pancreatitis promotes the generation of two different exosome populations.

Exosomes are small extracellular vesicles that act as intercellular messengers. Previous studies revealed that, during acute pancreatitis, circulating exosomes could reach the alveolar compartment and activate macrophages. However, proteomic analysis suggested that the most likely origin of these exosomes could be the liver instead of the pancreas. The present study aimed to characterize the exosomes released by pancreas to pancreatitis-associated ascitic fluid (PAAF) as well as those circulating in plasma in an experimental model of taurocholate-induced acute pancreatitis in rats. We provide evidence that during acute pancreatitis two different populations of exosomes are generated with relevant differences in cell distribution, protein and microRNA content as well as different implications in their physiological effects. During pancreatitis plasma exosomes, but not PAAF exosomes, are enriched in the inflammatory miR-155 and show low levels of miR-21 and miR-122. Mass spectrometry-based proteomic analysis showed that PAAF exosomes contains 10-30 fold higher loading of histones and ribosomal proteins compared to plasma exosomes. Finally, plasma exosomes have higher pro-inflammatory activity on macrophages than PAAF exosomes. These results confirm the generation of two different populations of exosomes during acute pancreatitis. Deep understanding of their specific functions will be necessary to use them as therapeutic targets at different stages of the disease.

Recurrent attacks of acute hepatic porphyria: major role of the chronic inflammatory response in the liver.

BACKGROUND: Acute intermittent porphyria (AIP) is an inherited disorder of haem metabolism characterized by life-threatening acute neurovisceral attacks due to the induction of hepatic δ-aminolevulinic acid synthase 1 (ALAS1) associated with hydroxymethylbilane synthase (HMBS) deficiency. So far, the treatment of choice is hemin which represses ALAS1. The main issue in the medical care of AIP patients is the occurrence of debilitating recurrent attacks. OBJECTIVE: The aim of this study was to determine whether chronic hemin administration contributes to the recurrence of acute attacks. METHODS: A follow-up study was conducted between 1974 and 2015 and included 602 French AIP patients, of whom 46 had recurrent AIP. Moreover, we studied the hepatic transcriptome, serum proteome, liver macrophage polarization and oxidative and inflammatory profiles of Hmbs-/- mice chronically treated by hemin and extended the investigations to five explanted livers from recurrent AIP patients. RESULTS: The introduction of hemin into the pharmacopeia has coincided with a 4.4-fold increase in the prevalence of chronic patients. Moreover, we showed that both in animal model and in human liver, frequent hemin infusions generate a chronic inflammatory hepatic disease which induces HO1 remotely to hemin treatment and maintains a high ALAS1 level responsible for recurrence. CONCLUSION: Altogether, this study has important impacts on AIP care underlying that hemin needs to be restricted to severe neurovisceral crisis and suggests that alternative treatment targeting the liver such as ALAS1 and HO1 inhibitors, and anti-inflammatory therapies should be considered in patients with recurrent AIP.

Spanish human proteome project: dissection of chromosome 16.

The Chromosome 16 Consortium forms part of the Human Proteome Project that aims to develop an entire map of the proteins encoded by the human genome following a chromosome-centric strategy (C-HPP) to make progress in the understanding of human biology in health and disease (B/D-HPP). A Spanish consortium of 16 laboratories was organized into five working groups: Protein/Antibody microarrays, protein expression and Peptide Standard, S/MRM, Protein Sequencing, Bioinformatics and Clinical healthcare, and Biobanking. The project is conceived on a multicenter configuration, assuming the standards and integration procedures already available in ProteoRed-ISCIII, which is encompassed within HUPO initiatives. The products of the 870 protein coding genes in chromosome 16 were analyzed in Jurkat T lymphocyte cells, MCF-7 epithelial cells, and the CCD18 fibroblast cell line as it is theoretically expected that most chromosome 16 protein coding genes are expressed in at least one of these. The transcriptome and proteome of these cell lines was studied using gene expression microarray and shotgun proteomics approaches, indicating an ample coverage of chromosome 16. With regard to the B/D section, the main research areas have been adopted and a biobanking initiative has been designed to optimize methods for sample collection, management, and storage under normalized conditions and to define QC standards. The general strategy of the Chr-16 HPP and the current state of the different initiatives are discussed.

The peptide-binding motif of HLA-DR8 shares important structural features with other type 1 diabetes-associated alleles.

The objective of this study was to characterize the peptide-binding motif of the major histocompatibility complex (MHC) class II HLA-DR8 molecule included in the type 1 diabetes-associated haplotype DRB1(*)0801-DQA1(*)0401/DQB1(*)0402 (DR8-DQ4), and compare it with that of other diabetes-associated MHC class II alleles; DR8-bound peptides were eluted from an HLA-DR homozygous lymphoblastoid cell line. The repertoire was characterized by peptide sequencing using a LTQ ion trap mass spectrometer coupled to a multidimensional liquid chromatography system. After validation of the spectra identification, the definition of the HLA-DR8 peptide-binding motif was achieved from the analysis of 486 natural ligands, based on serial alignments of all possible HLA-DR-binding cores. The DR8 motif showed a strong similarity with the peptide-binding motifs of other MHC class II diabetes-associated alleles, HLA-DQ8 and H-2 I-A(g7). Similar to HLA-DQ8 and H-2 I-A(g7), HLA-DR8 preferentially binds peptides with an acidic residue at position P9 of the binding core, indicating that DR8 is the susceptibility component of the DR8-DQ4 haplotype. Indeed, some DR8 peptides were identical to peptides previously identified as DQ8- or I-A(g7) ligands, and several diabetes-specific peptides associated with DQ8 or I-A(g7) could theoretically bind to HLA-DR8. These data further strengthen the association of HLA-DR8 with type I diabetes.

Landing differences between men and women in a maximal vertical jump aptitude test.

AIM: The aim of this study was to analyze the gender differences in the vertical ground reaction forces and the position of the center of gravity during the landing phase of a maximal vertical jump aptitude test. METHODS: The push-off, flight and landing phases of the jumps of 291 males (age = 19.6+/-2.8 years) and 92 females (age = 19.2+/-2.6 years), applicants to a Spanish faculty of sports sciences, were analyzed with a force platform. RESULTS: The greatest differences between men and women were found in the jump performance (women = 25.6+/-3.5 cm; men = 35.5+/-4.5 cm) and second peak vertical force value of the landing phase (women = 5.89+/-2.06 times body weight; men = 7.51 +/-2.38 times body weight), the values being greater in the men's group (P < 0.001). Correlation coefficients showed that the women utilized a different landing pattern than the one utilized by the men. CONCLUSION: Contrary to the authors' expectations, women showed lower second peak vertical force values during the landing. Taking into account only a kinetic point of view, they would have a lower risk of injury during the landing movement of maximal jumps. The lower values in the peak force, the delay of the impact of the calcaneus and the longer path of the center of gravity during the landing phase found in the women's group were related to a landing technique that is different from that of men.

Assessment of power output in jump tests for applicants to a sports sciences degree.

AIM: Our study aimed: 1) to describe the jump performance in a population of male applicants to a Faculty of Sports Sciences, 2) to apply different power equations from the literature to assess their accuracy, and 3) to develop a new regression equation from this population. METHODS: The push off phases of the counter-movement jumps (CMJ) on a force platform of 161 applicants (age: 19+/-2.9 years; weight: 70.4+/-8.3 kg) to a Spanish Faculty of Sports Sciences were recorded and subsequently analyzed. Their hands had to be placed on the hips and the knee angle during the counter movement was not controlled. Each subject had 2 trials to reach a minimum of 29 cm of jump height, and when 2 jumps were performed the best trial was analyzed. Multiple regression analysis was performed to develop a new regression equation. RESULTS: Mean jump height was 34.6+/-4.3 cm, peak vertical force 1 663.9+/-291.1 N and peak power 3524.4+/-562 W. All the equations underestimated power, from 74% (Lewis) to 8% (Sayers). However, there were high and significant correlations between peak power measured on the force platform, and those assessed by the equations. CONCLUSIONS: The results of the present study support the development of power equations for specific populations, to achieve more accurate assessments. The power equation from this study [Power = (62.5 x jump height (cm)) + (50.3 x body mass (kg)) 2184.7] can be used accurately in populations of male physical education students.

F2 isoprostane is already increased at the onset of type 1 diabetes mellitus: effect of glycemic control.

Much evidence has suggested that oxidative stress (OS) may play a role in the pathogenesis of diabetic complications. However, the relationship between hyperglycemia and OS is inconsistent in diabetic clinical studies. The aim of this study was to evaluate the effect of normalization of blood glucose levels on urinary 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)) excretion at the onset of type 1 diabetes. We studied 14 type 1 diabetic patients (50% males; mean age, 24.3 +/- 4.9 years) and 14 control subjects matched by age and body mass index. A 24-hour urine collection was performed to determine 8-epi-PGF(2alpha) as an integrated index of OS production at baseline, before starting insulin therapy, and 16 weeks later. Insulin treatment induced a significant reduction in glycosylated hemoglobin (HbA(1c)) (from 11.5% to 5.4% P =.0001), triglycerides (from 1.0 to 0.8 mmol/L, P =.002), and an increase in high-density lipoprotein (HDL)-cholesterol levels (from 1.1 to 1.5 nmol/L, P =.01) at week 16. This improvement in metabolic control was associated with a statistically significant reduction in 8-epi-PGF(2alpha) values (from 92.0 +/- 41.5 to 66.9 +/- 28.9 pg/mg urinary reatinine excretion, P =.015), although compared with the control group, 8-epi-PGF(2alpha) values remained higher in diabetic patients (66.9 +/- 28.9 v 39.1 +/- 13.8 pg/mg creatinine, P =.004). Enhanced OS is present in early clinical phases of type 1 diabetes, and the amelioration in metabolic control is associated with improvement in this pathogenic pathway.

Cigarette smoke concentrate increases 8-epi-PGF2alpha and TGFbeta1 secretion in rat mesangial cells.

Epidemiological studies have shown that cigarette smoke, an oxidant agent, is a risk factor for the development of diabetic nephropathy (DN), in which pathogenesis transforming growth factor beta(1) (TGFbeta(1)) plays a key role. In our experimental model we exposed mesangial cell cultures to cigarette smoke concentrate (CSC) to study the effect of smoking on the pathogenesis of DN. Thus, we analyzed the effect of CSC on TGFbeta(1) and lipid peroxidation (8-epi-PGF(2alpha)) in rat mesangial cells. Furthermore, since the protein kinase C (PKC) pathway appears to be a key factor for the enhanced production of TGFbeta(1), we also analyzed the effect of the selective PKCbeta inhibitor LY379196 on TGFbeta(1) response to CSC. CSC induced an increase of both TGFbeta(1) and 8-epi-PGF(2) compared to basal conditions (5 mM glucose). The CSC-induced increase in TGFbeta(1) secretion was significantly suppressed by LY379196. These data suggest that smoking could increase TGFbeta(1) production, probably due to oxidative stress and PKCbeta activation. This finding supports the concept that smoking is a risk factor for DN development.

The effects of smoking and its cessation on 8-epi-PGF2alpha and transforming growth factor-beta 1 in Type 1 diabetes mellitus.

BACKGROUND: Oxidative stress and transforming growth factor-beta 1 (TGF-beta1) are associated with diabetic complications, and smoking is a risk factor. AIMS: This study aimed (i) to compare urinary 8-epi-PGF2alpha and plasma and urinary TGF-beta1 levels obtained in heavy smokers with Type 1 diabetes with those observed in age-matched non-smoker patients with Type 1 diabetes and controls, and (ii) to investigate the effects of smoking cessation (SC) on the above-mentioned parameters in patients with Type 1 diabetes. METHODS AND RESULTS: Compared with control subjects (n = 12), non-smoker diabetic patients (n = 12) presented higher values of urinary 8-epi-PGF2alpha (74.2 +/- 29.6 vs. 29.6 +/- 11.1 pg/mg urinary creatinine, P = 0.01), plasma TGF-beta1 (7.7 +/- 4.7 vs. 3.6 +/- 1.7 ng/ml, P = 0.001) and urinary TGF-beta1 (15.3 +/- 6.3 vs. 8.1 +/- 4.4 ng/mg urinary creatinine, P = 0.02). Compared with non-smoker diabetic patients, smoker diabetic patients (n = 16) showed higher levels of urinary 8-epi-PGF2alpha (107.8 +/- 40.2 vs. 74.2 +/- 29.6 pg/mg urinary creatinine, P = 0.0001), plasma TGF-beta1 (12.6 +/- 4.9 vs. 7.7 +/- 4.7 ng/ml, P = 0.001) and urinary TGF-beta1 (27.5 +/- 16.0 vs. 15.3 +/- 6.3 ng/mg urinary creatinine, P = 0.01). Smoker patients were included in a smoking cessation programme. In the 10 patients that gave up smoking there was a reduction of urinary 8-epi-PGF2alpha (basal: 110.47 +/- 47.0 vs. week 12: 73.2 +/- 25.6; P < 0.001), plasma TGF-beta1 (basal: 11.2 +/- 5.9 vs. week 12: 4.89 +/- 2.25; P < 0.01) and urinary TGF-beta1 (basal: 18.12 +/- 9.27 vs. week 12: 10.32 +/- 2.0; P < 0.01) levels. CONCLUSIONS: In patients with Type 1 diabetes, smoking increased oxidative stress, evaluated by lipid peroxidation, and TGF-beta1 production. Smoking cessation decreased these parameters, providing additional support to encourage diabetic patients to give up smoking.

Biochemical characterization of recombinant and circulating human Spalpha.

Human Spalpha is a soluble protein expressed by macrophages present in lymphoid tissues (spleen, lymph node, thymus, and bone marrow), for which little functional and structural information is available. It belongs to the group B of the scavenger receptor cysteine-rich superfamily (SRCR-SF) that includes the lymphocyte surface receptors CD5 and CD6 among others. Spalpha is able to bind to different cells of the immune system (monocytes and lymphocytes), which suggests that it may play an important role in the regulation of this system. To study Spalpha, an episomal mammalian expression system (pCEP-Pu/HEK 293-EBNA) was used to produce a recombinant form (rSpalpha) that was utilized for biochemical studies and for the generation of specific hybridomas. Four monoclonal antibodies were selected for their reactivity against rSpalpha by Western blot, immunoprecipitation, and enzyme-linked immunosorbent assays. The monoclonal antibodies recognized three different epitopes on Spalpha. The monoclonal antibodies revealed the existence of two Spalpha isoforms of 38 and 40 kDa, resulting from different sialic acid content. They also showed that Spalpha is a relatively abundant serum protein (60 micro g/ml) that mostly circulates in association with other serum proteins. Accordingly, rSpalpha allowed affinity chromatography isolation of polyclonal and monoclonal immunoglobulin M (IgM). These data indicate that Spalpha is a circulating protein that may play a role in the homeostasis of IgM antibodies.

Nuclear ploidy is contingent on the microtubular cycle responsible for plant cytokinesis.

Division of the plant cell relies on the preprophase band of microtubules (PPB)-phragmoplast system. Cells of onion (Allium cepa L.) root meristems were rendered binucleate by preventing the consolidation of cell plate formation in telophase with 5 mM caffeine. These binucleates developed either a single PPB around one of their two nuclei or two PPBs, one per nucleus, in the prophase of the ensuing mitosis. Prophase cells developing one single PPB were shorter in length (42.3 +/- 4.1 microm) than those developing 2 PPBs (49.8 +/- 4.1 microm), and interphase duration was inversely related to cell length. Cells whose length was less than or equal to 42 microm, i.e., which had not even reached the mean size of the small binucleates in prophase, were followed throughout mitosis. In metaphase, they always assembled two mitotic spindles (one per nucleus). However, the cells that had assembled a single PPB also developed a single phragmoplast in telophase, leading to polyploidization. As these meristematic cells were not wide enough to accommodate the midzones of both mitotic spindles in any single plane transversal to the cell elongation axis, the spindles tilted until their midzones formed a continuum where the single common phragmoplast assembled. Its position was thereby uncoupled from that of the preceding PPB. Subsequently, the chromosomes from two different half-spindles were included, by a common nuclear envelope, in a single tetraploid nucleus. Finally, the cytokinetic plate segregated the two tetraploid nuclei formed at each side of the phragmoplast into two independent sister cells.

G2 checkpoint targets late replicating DNA.

In the multinucleate cells induced in Allium cepa L. meristems, the nuclei surrounded by the largest cytoplasm environment complete replication earlier (advanced nuclei), but have a longer G2, than the others (delayed nuclei). Thus, all nuclei break down the nuclear envelope and start metaphase simultaneously. The present report shows that this synchronization relies on a checkpoint mechanism. When completion of replication was prevented in the delayed nuclei (due to in vivo 5-aminouracil feeding initiated when the advanced nuclei were already in G2), the metaphase was also further delayed in the advanced ones. In turn, some of the delayed nuclei overrode the G2 checkpoint (adaptation) and entered into mitosis with broken chromatids (Del Campo et al., 1997). Anoxic UVA (313 nm) irradiation apparently prevents the binding of regulatory proteins to Br-DNA. The present report shows that late replicating sequences are the targets of the checkpoint signal produced by the still replicating nuclei. This signal delays metaphase in the advanced nuclei, whose DNA is already fully replicated. Thus, when the already replicated sequences of late replicating DNA was modified in the advanced nuclei by bromosubstitution followed by anoxic UVA irradiation, they entered into mitosis without any delay, ignoring the inhibitory signals produced by the still replicating nuclei.

Preparation of porous n-type silicon sample plates for desorption/ionization on silicon mass spectrometry (DIOS-MS).

This study focuses on porous silicon (pSi) fabrication methods and properties for desorption ionization on silicon mass spectrometry (DIOS-MS). PSi was prepared using electrochemical etching of n-type silicon in HF-ethanol solution. Porous areas were defined by a double-sided illumination arrangement: front-side porous areas were masked by a stencil mask, eliminating the need for standard photolithography, and backside illumination was used for the backside ohmic contact. Backside illumination improved the uniformity of the porosified areas. Porosification conditions, surface derivatizations and storage conditions were explored to optimize pSi area, pore size and pore depth. Chemical derivatization of the pSi surfaces improved the DIOS-MS performance providing better ionization efficiency and signal stability with lower laser energy. Droplet spreading and drying patterns on pSi were also examined. Pore sizes of 50-200 nm were found to be optimal for droplet evaporation and pore filling with the sample liquid, as measured by DIOS efficiency. With DIOS, significantly better detection sensitivity was obtained (e.g. 150 fmol for midazolam) than with desorption ionization from a standard MALDI steel plate without matrix addition (30 pmol for midazolam). Also the noise that disturbs the detection of low-molecular weight compounds at m/z < 500 with MALDI could be clearly reduced with DIOS. Low background MS spectra and good detection sensitivity at the 100-150 fmol level for pharmaceutical compounds were achieved with DIOS-MS.

Automated strong cation exchange extraction of fatty acid esters of 3-(N-phenylamino)-1,2-propanediol from oil samples for routine quantification by HPLC-APCI/MS/MS.

Fatty acid esters of 3-(N-phenylamino)-1,2-propanediol are currently considered the best chemical markers of toxic oils related to the Spanish toxic oil syndrome. Recent research in this area has undertaken the exhaustive and quantitative characterization of these compounds in oils collected during the epidemic outbreak. Current methods developed in this laboratory are based on solid phase extraction (SPE) using SCX cartridges followed by HPLC-APCI/MS/MS quantification. To circumvent the long and tedious extraction procedure, the SPE protocol was adapted for automatic extraction and the problems derived from the use of the immiscible solvents required for the SCX extraction were solved. Linearity of the analytical method was found in the same range as for the manual method. Extraction recoveries were 87 and 75% for 2-hydroxy-3-(N-phenylamino)propyl linoleate and 2-(linoleyloxy)-3-(N-phenylamino)propyl linoleate, respectively, and the corresponding coefficients of variation were approximately 1%, greatly improving reproducibility over manual procedures.

Determination of aniline derivatives in oils related to the toxic oil syndrome by atmospheric pressure ionization-tandem mass spectrometry.

In 1981, an unknown disease appeared in Spain, the Spanish Toxic Oil Syndrome. Nowadays and despite all efforts, the etiological agent is still unknown. Early studies showed a link between this illness and the consumption of denatured rapeseed oil fraudulently processed and marketed as edible oil. Two families of aniline derivatives present in these oils (fatty acid anilides and acylated phenyl amino propanediol derivatives or PAPs) were found to be good chemical markers of toxic oils. In this work, a new method has been developed to analyze these aniline derivatives in oil samples by HPLC-MS and HPLC-MS/MS with an API source. For their quantification, three different internal standards were used, one for anilides and two for PAPs. Quantification limits were 8 ppm for anilides and 0.2 ppm for PAPs. Anilides and PAPs were found in marker-positive samples at levels up to 50,000 and 330 ppm, respectively. The relative abundance of the different fatty acid anilides and PAPs correlates with the fatty acid composition of the oils. More than 2,600 different samples were analyzed by this method in the most exhaustive screening of suspected toxic oils carried out to date.

Storage time and deodorization temperature influence the formation of aniline-derived compounds in denatured rapeseed oils.

In 1981 an epidemic, named Toxic Oil Syndrome, occurred in Spain as a result of ingestion of rapeseed oil denatured with 2% aniline, which had been imported for industrial use but was fraudulently diverted and processed for human consumption. Two groups of chemical compounds have been identified in the ingested toxic oil: fatty acid anilides and amino-propanediol derivatives. The objective of this work was to assess the effect of several refining process variables on the formation of 3-(N-phenylamino)-1,2-propanediol (PAP) esters. The amount of PAP esters in aniline-denatured oil increased dramatically when oil was heated from 250 degrees C to 300 degrees C. However, the ones formed when 300 degrees C was reached were lost during processing at that temperature. The level maintained during the operation time at 300 degrees C was higher in denatured samples stored for 3 weeks before refining than in denatured samples stored only for 1 week. Anilides were also analyzed. We found that anilides decreased very little with distillation time. In this paper we discuss the influence of storage time prior to refining and of elevated refining temperature, such as temperatures that might occur in close proximity to a deodorizer coil.

Synchronous nuclear-envelope breakdown and anaphase onset in plant multinucleate cells.

Multinucleate plant cells with genetically balanced nuclei can be generated by inhibiting cytokinesis in sequential telophases. These cells can be used to relate the effect of changes in the distribution of nuclei in the cytoplasm to the control of the timing of cell cycle transitions. Which mitotic cell cycle events are sensitive to differences in the amount of cytoplasm surrounding each chromosomal complement has not been determined. To address this, we maximized the cell size by transiently inhibiting replication, while cell growth was not affected. The nuclei of 93% of the elongated cells reached prophase asynchronously compared to 46% of normal-sized multinucleate cells. The asynchronous prophases of normal-sized cells became synchronous at the time of nuclear-envelope breakdown, and the ensuing metaphase plate formation and anaphase onset and progression occurred synchronously. The elongated multinucleate cells were also very efficient in synchronizing the prophases at nuclear-envelope breakdown, in the prophase-to-prometaphase transition. However, 2.4% of these cells broke down the nuclear envelope asynchronously, though they became synchronous at the metaphase-to-anaphase transition. The kinetochore-microtubular cycle, responsible for coordinating the metaphase-to-anaphase transition and for the rate of sister segregation to opposite spindle poles during anaphase, remained strictly controlled and synchronous in the different mitoses of a single cell, independently of differences in the amount of cytoplasm surrounding each mitosis or its ploidy. Moreover, the degree of chromosome condensation varied considerably within the different mitotic spindles, being higher in the mitoses with the largest surrounding cytoplasm.

Absorption and effects of 3-(N-phenylamino)-1,2-propanediol esters in relation to toxic oil syndrome.

Toxic Oil Syndrome (TOS) was an epidemic disease related to the consumption of rapeseed oil denatured with aniline that made its sudden appearance in Spain in 1981. The fatty acid esters of 3-(N-phenylamino)-1,2-propanediol (PAP), which is a chemical class of by-products resulting from the reaction of aniline with oil components, have shown a strong association with TOS-related oils. These compounds also show some structural similarities to platelet-activating factor (PAF). In search of a toxic agent that could explain the widespread systemic effects observed in TOS patients, we investigated the intestinal absorption and biotransformation of the different PAP esters found in TOS-related oil samples and the possible pathophysiological effect of these mediators and their metabolic products if acting as PAF analogs. Results indicate that PAP esters are absorbed in the gastrointestinal tract and are distributed and stored in different organs, particularly in the liver and brown adipose tissue. PAP in these organs showed different patterns of fatty acids, indicating the ability of the gastrointestinal tract to modify the fatty acid composition of the parent PAP. Thus, the fatty acid profile of the PAP esters found in intestine appears to be related to the type of oil used as vehicle. Some of these PAP esters, when a long acyl chain was present in the sn-1 position of the molecule, showed an inhibitory effect on the PAF synthesis. This is an important observation in line with the systemic nature of the disease.

Premitotic chromosome individualization in mammalian cells depends on topoisomerase II activity.

When DNA topoisomerase II (topo II) activity is inhibited with a non-DNA-damaging topo II inhibitor (ICRF-193), mammalian cells become checkpoint arrested in G2-phase. In this study, we analyzed chromosome structure in cells that bypassed this checkpoint. We observed a novel type of chromosome aberration, which we call omega-figures. These are entangled chromosome regions that indicate the persistence of catenations between nonhomologous sequences. The number of omega-figures per cell increased sharply as cells evaded the transient block imposed by the topo II-dependent checkpoint, and the presence of caffeine (a checkpoint-evading agent) potentiated this increase. Thus, the removal of nonreplicative catenations, a process that promotes chromosome individualization in G2, may be monitored by the topo II-dependent checkpoint in mammals.