Patulin in grape must and new, still fermenting wine (Federweißer).

Extreme differences in temperature were anticipating a high mycotoxin contamination of the 2006 vintage. 96 samples of different grapes and must qualities of the producing region Mosel-Saar-Ruwer as well as Federweißer from retail sales were analyzed for patulin according to EN 14177, whereat the fermentation of the must was stopped by addition of sodium azide.

T-2 and HT-2 toxin analysis in cereals and cereal products following IAC cleanup and determination via GC-ECD after derivatization.

The authors present a new and sensitive method for the determination of T-2- und HT-2 Toxin in cereals and cereal products in the low ppb level. A representative part of the cereal sample is extracted with a mixture of methanol-water (90:10) and the extract is cleaned on the commercially available immunoaffinity column T-2test™ (IAC), eluted with methanol, derivatized by pentafluorpropionic anhydride (PFPA) and measured on a GC-ECD. The method has been successfully validated on wheat, rye and oats. The recovery rates with wheat and rye endowed on a level of 50 ppb and with 85 ppb naturally contaminated oats were 71-115% with a coefficient of variation of 5.7-19.5%. The detection limits of the method with a signal to noise level of 3:1 were 1.5-2.3 µg/kg for HT-2 and 1.1-1.7 µg/kg for T-2 toxin.

Fumonisin intake of the German consumer.

In order to calculate the dietary fumonisin intake of the German consumer, a large survey was carried out on a variety of potentially contaminated products in the period between December 1998 and July 2001. A total of 1960 food samples comprising all known relevant groups of products were analysed for fumonisins. Furthermore, 272 of these samples were also analysed for hydrolysed fumonisins (HFB). For routine analysis enzyme immunoassay was used, confirmatory and control analyses were performed using HPLC-FLD after precolumn derivatisation, or by LC-MS/MS. Daily intake of fumonisins was calculated by combining fumonisin contamination data obtained in this study with available food consumption data for Germany. In a "mean case" scenario, median fumonisin levels in foods and mean food intake values were used. To generate a "bad case" scenario, the 90(th) percentile of fumonisin levels in foods and mean food intake values were combined. The overall daily fumonisin intake by the German consumer was 1.1 µg in the "mean case" scenario, and 21 µg in the "bad case" scenario. It was concluded that in general there is no increased risk for the German consumer in aspects of exceeding the recommended tolerable daily intake of fumonisins (2 µg/kg body weight). However, certain products (and certain brands of products) were repeatedly found to contain elevated fumonisin levels, which in a "worst case" scenario ("high" food intake of maize-based products) could pose a potential risk for the consumer, in particular concerning foods for infants and young children. High fumonisin levels were found in infant foods in 1999, but contamination levels decreased strongly in the following years. HFBs (mostly HFB1) were frequently found in processed cereals such as corn flakes, but in relatively low concentrations. According to our findings, the new European Union maximum levels for fumonisins are suitable to eliminate peak contamination levels of fumonisins in foods, but would lead to a regular excess of the TDI for infants and young children if these maximum levels would indeed be exhausted.

[Not Available]. / Deoxynivalenol in lebensmitteln: Ergebnisse einer Pilotstudie.

Cereal food products (n=333) were purchased in retail stores from Germany in 2001 and analysed for deoxynivalenol (DON), either by enzyme immunoassay or by HPLC after immunoaffinity chromatographic cleanup. Detection limits were dependent of the sample matrix and varied from 20-100 µg/kg. The overall DON incidence was 53%, with mean and median levels for positives of 251 µg/kg and 142 µg/kg, respectively. The contamination with DON (mean/median value, µg/kg) as found for bread (90/87), wheat flour (161/124), and noodles (472/297) indicate that the levels of DON in cereal foods were significant in view of the tolerable daily intake (1 µg/kg body weight) as established by the European Union scientific committee on food.

[Not Available]. / Aufreinigung von Fumonisinen und ihren Hydrolysaten über ein neuartiges Festphasenextraktions-Material.

Tests with various clean-up materials after optimisation of different parameters showed that the use of Oasis® material resulted in matrixless chromatograms in HPLC-FLD. The selectivity and detection limit of the method was improved by using LC-MS/MS as the detection system. Mean recovery was 100%, and no negative food matrix effects could be observed.

Inositol 1,3,4-trisphosphate 5/6-kinase is a protein kinase that phosphorylates the transcription factors c-Jun and ATF-2.

Phosphorylation of inositol 1,3,4-trisphosphate by inositol 1,3,4-trisphosphate 5/6-kinase is the first committed step in the formation of higher phosphorylated forms of inositol. We have shown that the eight proteins called the COP9 signalosome complex copurify with calf brain 5/6-kinase. Because the complex has been shown to phosphorylate c-Jun in vitro, we tested both the complex and 5/6-kinase and found that both are able to phosphorylate c-Jun and ATF-2 on serine/threonine residues. These findings establish a link between two major signal transduction systems: the inositol phosphates and the stress response system.

Characterization of an adapter subunit to a phosphatidylinositol (3)P 3-phosphatase: identification of a myotubularin-related protein lacking catalytic activity.

The D3-phosphoinositides act as second messengers by recruiting, and thereby activating, diverse signaling proteins. We have previously described the purification of a rat phosphatidylinositol 3-phosphate [PtdIns(3)P] 3-phosphatase, comprising a heterodimer of a 78-kDa adapter subunit in complex with a 65-kDa catalytic subunit. Here, we have cloned and characterized the cDNA encoding the human 3-phosphatase adapter subunit (3-PAP). Sequence alignment showed that 3-PAP shares significant sequence similarity with the protein and lipid 3-phosphatase myotubularin, and with several other members of the myotubularin gene family including SET-binding factor 1. However, unlike myotubularin, 3-PAP does not contain a consensus HCX(5)R catalytic motif. The 3-PAP sequence contains several motifs that predict interaction with proteins containing Src homology-2 (SH2) domains, phosphotyrosine-binding (PTB) domains, members of the 14-3-3 family, as well as proteins with SET domains. Northern blot analysis identified two transcripts (5.5 kb and 2.5 kb) with highest abundance in human liver, kidney, lung, and placenta. 3-PAP immunoprecipitates isolated from platelet cytosol hydrolyzed the D3-phosphate from PtdIns(3)P and PtdIns 3,4-bisphosphate [PtdIns(3,4)P(2)]. However, insect cell-expressed 3-PAP recombinant protein was catalytically inactive, confirming our prior prediction that this polypeptide represents an adapter subunit.

Ochratoxin A (OTA) in coffee: nation-wide evaluation of data collected by German Food Control 1995-1999.

The evaluation process involved data collected by Official Food Control Laboratories during the period 1995 until 1999. A total of 613 samples analysed for ochratoxin A and complying with a detection limit lower than 0.6 microg/kg were evaluated. With the assistance of statistical process analysis the median concentrations for green coffee (0.4 microg/kg), for roasted coffee (0.6 microg/kg), for decaffeinated roasted coffee along with low-acid decaffeinated roasted coffee (0.4 microg/kg) as well as for soluble coffee (0.7 microg/kg) were determined. The result is a mean daily total intake per consumer of 9 ng OTA.

Overexpression of the inositol phosphatase SopB in human 293 cells stimulates cellular chloride influx and inhibits nuclear mRNA export.

SopB is an inositol phosphate phosphatase that is a virulence factor in Salmonella species. We have overexpressed SopB cDNA in a tetracycline-dependent system in human embryonic 293 cells, and used this model system to directly analyze the role of SopB in altering inositol metabolite levels in vivo. Addition of tetracycline to these cells resulted in the rapid induction of SopB expression, which was coincident with perturbations in the cellular levels of multiple soluble inositol phosphates. All of the changes induced by SopB expression were reversed within 24 h on removal of tetracycline from media. Specifically, cellular inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) and inositol hexakisphosphate (InsP(6)) levels were depleted within 4 to 6 h after inducing SopB expression. A transient rise in cellular inositol 1,4,5,6-tetrakisphosphate was also observed and was accompanied by increased chloride channel activity. This indicates that SopB alone is sufficient for changes in chloride channel function in cells infected with Salmonella organisms. Depletion of inositol phosphates, including InsP(5) and InsP(6) metabolites, was coincident with the accumulation of polyadenylated RNA in the nucleus. This suggested that a defect in nuclear export had occurred. Moreover, the penetrance of the export defect required localization of SopB to the nucleus. These results provide evidence that inositol phosphate productions may be required for efficient mRNA export in mammalian cells.

Inositol polyphosphate 4-phosphatase type I regulates cell growth downstream of transcription factor GATA-1.

Megakaryocytes lacking transcription factor GATA-1 fail to complete maturation in vivo and hyperproliferate. To define how GATA-1 regulates megakaryocyte cell growth we searched for mRNA transcripts expressed in primary wild-type, but not GATA-1(-), megakaryocytes. One differentially expressed transcript encodes inositol polyphosphate 4-phosphatase type I (4-Ptase I). This enzyme hydrolyses phosphatidylinositol 3,4-bisphosphate and also has lesser activity against soluble analogues of this lipid, inositol 3, 4-bisphosphate and inositol 1,3,4-triphosphate. Reintroduction of 4-Ptase I into both primary GATA-1(-) and wild-type megakaryocytes significantly retards cell growth, suggesting that absence of 4-Ptase I may contribute to the hyperproliferative phenotype of GATA-1(-) megakaryocytes. Overexpression of 4-Ptase I also markedly reduces growth of NIH 3T3 fibroblasts. Taken together, these data indicate that 4-Ptase I is a regulator of cell proliferation.

Occurrence of ochratoxin A (OTA) in wines: influence of the type of wine and its geographical origin.

The results of more than 450 samples taken from the literature and 400 samples tested by our own investigations have been taken into account to quite extensively describe the situation of OTA contamination of wine. According to these data OTA is much more commonly detected in red wines than in rosé and white wines and the OTA concentration is remarkably higher than in the last two. Thus OTA could be detected in 25% of white wine samples whereas it was detected in 40% of rosé and 54% of red wine samples. The same result was found when comparing the wines from southern and northern regions. Here the red wine samples from the northern cultivating area showed a contamination of 12% in contrast to those from the southern area which showed a contamination of about 95%.

The isolation and characterization of a cDNA encoding phospholipid-specific inositol polyphosphate 5-phosphatase.

We report the cDNA cloning and characterization of a novel human inositol polyphosphate 5-phosphatase (5-phosphatase) that has substrate specificity unlike previously described members of this large gene family. All previously described members hydrolyze water soluble inositol phosphates. This enzyme hydrolyzes only lipid substrates, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate. The cDNA isolated comprises 3110 base pairs and predicts a protein product of 644 amino acids and M(r) = 70,023. We designate this 5-phosphatase as type IV. It is a highly basic protein (pI = 8.8) and has the greatest affinity toward phosphatidylinositol 3,4,5-trisphosphate of known 5-phosphatases. The K(m) is 0.65 micrometer, 1/10 that of SHIP (5.95 micrometer), another 5-phosphatase that hydrolyzes phosphatidylinositol 3,4,5-trisphosphate. The activity of 5-phosphatase type IV is sensitive to the presence of detergents in the in vitro assay. Thus the enzyme hydrolyzes lipid substrates in the absence of detergents or in the presence of n-octyl beta-glucopyranoside or Triton X-100, but not in the presence of cetyltriethylammonium bromide, the detergent that has been used in other studies of the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Remarkably SHIP, a 5-phosphatase previously characterized as hydrolyzing only substrates with d-3 phosphates, also readily hydrolyzed phosphatidylinositol 4,5-bisphosphate in the presence of n-octyl beta-glucopyranoside but not cetyltriethylammonium bromide. We used antibodies prepared against a peptide predicted by the cDNA to identify the 5-phosphatase type IV enzyme in human tissues and find that it is highly expressed in the brain as determined by Western blotting. We also performed Western blotting of mouse tissues and found high levels of expression in the brain, testes, and heart with lower levels of expression in other tissues. mRNA was detected in many tissues and cell lines as determined by Northern blotting.

The activation loop of phosphatidylinositol phosphate kinases determines signaling specificity.

Phosphatidylinositol-4,5-bisphosphate plays a pivotal role in the regulation of cell proliferation and survival, cytoskeletal reorganization, and membrane trafficking. However, little is known about the temporal and spatial regulation of its synthesis. Higher eukaryotic cells have the potential to use two distinct pathways for the generation of phosphatidylinositol-4,5-bisphosphate. These pathways require two classes of phosphatidylinositol phosphate kinases, termed type I and type II PIP kinases. While highly related by sequence, these kinases localize to different subcellular compartments, phosphorylate distinct substrates, and are functionally nonredundant. Here, we show that a 20- to 25-amino acid loop spanning the catalytic site, termed the activation loop, determines both enzymatic specificity and subcellular targeting of PIP kinases. Therefore, the activation loop controls signaling specificity and PIP kinase function at multiple levels.

[Not Available]. / Bestimmung von Fumonisinen in komplexen Lebensmittelmatrices mittels Accelerated Solvent Extraktion (ASE).

Accelerated solvent extraction (ASE) is an alternative sample extraction procedure for fumonisins in corn and corn products. ASE gave results comparable to that of a draft CEN method, but required less extraction time. Furthermore, ASE gave significantly higher quantitative values than another method reported for extraction of fumonisins (Trucksess et al., 1995).

Increased levels of plasma lysosomal enzymes in patients with Lowe syndrome.

Lowe syndrome is an X-linked disorder that has a complex phenotype that includes progressive renal failure and blindness. The disease is caused by mutations in an inositol polyphosphate 5-phosphatase designated OCRL. It has been shown that the OCRL protein is found on the surface of lysosomes and that a renal tubular cell line deficient in OCRL accumulated substrate phosphatidylinositol 4, 5-bisphosphate. Because this lipid is required for vesicle trafficking from lysosomes, we postulate that there is a defect in lysosomal enzyme trafficking in patients with Lowe syndrome that leads to increased extracellular lysosomal enzymes and might lead to tissue damage and contribute to the pathogenesis of the disease. We have measured seven lysosomal enzymes in the plasma of 15 patients with Lowe syndrome and 15 age-matched male controls. We find a 1.6- to 2.0-fold increase in all of the enzymes measured. When the data was analyzed by quintiles of activity for all of the enzymes, we found that 95% of values in the lowest quintile come from normal subjects whereas in the highest quintile 85% of the values are from patients with Lowe syndrome. The increased enzyme levels are not attributable to renal insufficiency because there was no difference in enzyme activity in the four patients with the highest creatinine levels compared with the six patients with the lowest creatinine values.

The inositol polyphosphate 4-phosphatase forms a complex with phosphatidylinositol 3-kinase in human platelet cytosol.

Inositol polyphosphate 4-phosphatase (4-phosphatase) is an enzyme that catalyses the hydrolysis of the 4-position phosphate from phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. In human platelets the formation of this phosphatidylinositol, by the actions of phosphatidylinositol 3-kinase (PI 3-kinase), correlates with irreversible platelet aggregation. We have shown previously that a phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase forms a complex with the p85 subunit of PI 3-kinase. In this study we investigated whether PI 3-kinase also forms a complex with the 4-phosphatase in human platelets. Immunoprecipitates of the p85 subunit of PI 3-kinase from human platelet cytosol contained 4-phosphatase enzyme activity and a 104-kDa polypeptide recognized by specific 4-phosphatase antibodies. Similarly, immunoprecipitates made using 4-phosphatase-specific antibodies contained PI 3-kinase enzyme activity and an 85-kDa polypeptide recognized by antibodies to the p85 adapter subunit of PI 3-kinase. After thrombin activation, the 4-phosphatase translocated to the actin cytoskeleton along with PI 3-kinase in an integrin- and aggregation-dependent manner. The majority of the PI 3-kinase/4-phosphatase complex (75%) remained in the cytosolic fraction. We propose that the complex formed between the two enzymes serves to localize the 4-phosphatase to sites of PtdIns(3,4)P2 production.

SopB, a protein required for virulence of Salmonella dublin, is an inositol phosphate phosphatase.

Several proteins secreted by enteric bacteria are thought to contribute to virulence by disturbing the signal transduction of infected cells. Here, we report that SopB, a protein secreted by Salmonella dublin, has sequence homology to mammalian inositol polyphosphate 4-phosphatases and that recombinant SopB has inositol phosphate phosphatase activity in vitro. SopB hydrolyzes phosphatidylinositol 3,4,5-trisphosphate, an inhibitor of Ca2+-dependent chloride secretion. In addition, SopB hydrolyzes inositol 1,3,4,5,6 pentakisphosphate to yield inositol 1,4,5, 6-tetrakisphosphate, a signaling molecule that increases chloride secretion indirectly by antagonizing the inhibition of chloride secretion by phosphatidylinositol 3,4,5-trisphosphate [Eckmann, L., Rudolf, M. T., Ptasznik, A., Schultz, C., Jiang, T., Wolfson, N., Tsien, R., Fierer, J., Shears, S. B., Kagnoff, M. F., et al. (1997) Proc. Natl. Acad. Sci. USA 94, 14456-14460]. Mutation of a conserved cysteine that abolishes phosphatase activity of SopB results in a mutant strain, S. dublin SB c/s, with decreased ability to induce fluid secretion in infected calf intestine loops. Moreover, HeLa cells infected with S. dublin SB c/s do not accumulate high levels of inositol 1,4,5,6-tetrakisphosphate that are characteristic of wild-type S. dublin-infected cells. Therefore, SopB mediates virulence by interdicting inositol phosphate signaling pathways.