Quantification of lupine (Lupinus angustifolius) in wheat flour using real-time PCR and an internal standard material

In the European Union, labelling of the 14 food allergens listed in Annex IIIa of Directive 2000/13/EC is mandatory. The implementation of upper limits for these allergens is under discussion. Therefore, quantitative analytical methods will be needed to verify compliance with regulatory requirements and to provide an improved basis for the legal assessment of allergen labelling. In this study, the lupine flour content in wheat flours was determined using real-time PCR and statice seeds as internal standard material. The method proved to be applicable to the quantification of lupine contents from 1 to 10?mg/kg, which is in the range relevant for allergic consumers.     

Validation of a Duplex Real-Time PCR for the Detection of Salmonella spp. in Different Food Products

A 5′ nuclease duplex real-time polymerase chain reaction (PCR) assay was developed and validated with various food products for the specific and fast detection of Salmonella spp. in food. The assay used previously published primers in combination with a newly developed probe targeting the invA gene. An internal amplification control, which is coamplified in a duplex PCR, was included in the assay. The analysis of 1,934 natural food samples with real-time PCR and the cultural method in parallel resulted in a relative accuracy of 100% and 99.84% respectively, depending on the enrichment procedure in which buffered peptone water and selective enrichment in Rappaport–Vassiliadis (RV) broth were employed. The duplex real-time PCR assay has proven to be a specific, sensitive and fast screening method for Salmonella spp. in food. The overall analysis time of the PCR method was approximately 28 h, in contrast to 4 to 5 days with conventional Salmonella diagnostics. The developed assay has been shown to be a reliable diagnostic tool for use in routine analysis. It has been validated thoroughly and has become an official method in Germany for the detection of Salmonella spp. in food.     

Calcium methoxide initiated ring-opening polymerization of ε-caprolactone and L-lactide

Summary A commercial calcium dimethoxide and an in-situ generated calcium methoxide prepared from bis(tetrahydrofuran)calcium bis[bis(trimethylsilyl)amide] and methanol, were investigated as initiators for the ring-opening polymerization of ε-caprolactone and L-lactide. Commercial calcium dimethoxide initiated rapid ε-caprolactone polymerization at 120°C in bulk to give quantitatively a polymer with a polydispersity index around 1.3. Significant racemization was observed for L-lactide polymerization. The In-situ formed calcium methoxide promoted the solution polymerization of both ε-caprolactone and L-lactide to high conversion at room temperature over a short time period, yielding the corresponding polyesters with narrow molecular weight distribution. NMR spectra showed that the poly(L-lactide) isolated had a purely isotactic microstructure. The initiator efficiency could be tuned by varying the molar ratio of methanol and bis(tetrahydrofuran)calcium bis[bis(trimethylsilyl)amide]. Received: 11 August 2000/Revised version: 21 December 2000/Accepted: 3 January 2001     

Säureabbau von Stärke unter semi-dry Bedingungen

Acid Modification of Starch in a Semi-dry Process . The semi-dry starch-modification process involves spraying the granular substance, which has been placed in a mixer, with acid in a catalytic concentration, after which it is heated briefly in a microwave field at a predetermined setting. The substance is then neutralized by spraying it with a soda solution and simultaneously cooled. During the mild acid hydrolysis, nonspecifically glycosidic bonds of the glucose polymers are disrupted, the amylopectin fraction first being affected to a greater extent than the amylose. By varying the modification conditions, in particular the quantity of catalyst used and the heating conditions, it was possible to produce not only a number of thin boiling starches with various degrees of gel formation but also dextrins which can be used as fat replacers. The semi-dry process has major advantages when compared with the slurry process: fewer chemicals are used, the reaction time is shorter, washing is not required and there is no waste water or loss of substance.     

Na+-Coupled Respiration and Reshaping of Extracellular Polysaccharide Layer Counteract Monensin-Induced Cation Permeability in Prevotella bryantii B14

Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B₁4 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P. bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P. bryantii B₁4 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na⁺-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P. bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.     

Nucleocapsid protein zinc-finger mutants of simian immunodeficiency virus strain mne produce virions that are replication defective in vitro and in vivo

All retroviruses (except the spumaretroviruses) contain a nucleocapsid (NC) protein that encodes one or two copies of the Zn2+-finger sequence -Cys-X2-Cys-X4-His-X4-Cys-. This region has been shown to be essential for recognition and packaging of the genomic RNA during virion particle assembly. Additionally, this region has been shown to be involved in early infection events in a wide spectrum of retroviruses, including mammalian type C [e.g., murine leukemia virus (MuLV)], human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus, and other retroviruses. Mutations in the two Zn2+-fingers of the NC protein of simian immunodeficiency virus strain Mne [SIV(Mne)] have been generated. The resulting virions contained the normal complement of processed viral proteins with densities indistinguishable from wild-type SIV(Mne). All of the mutants had electron micrograph morphologies similar to those of immature particles observed in wild-type preparations. RNA packaging was less affected by mutations in the NC protein of SIV(Mne) than has been observed for similar mutants in the MuLV and HIV-1 systems. Nevertheless, in vitro replication of SIV(Mne) NC mutants was impaired to levels comparable to those observed for MuLV and HIV-1 NC mutants; replication defective NC mutants are typically 10(5)- to 10(6)-fold less infectious than similar levels of wild-type virus. One mutant, DeltaCys33-Cys36, was also found to be noninfectious in vivo when mutant virus was administered intravenously to a pig-tailed macaque. NC mutations can therefore be used to generate replication defective virions for candidate vaccines in the SIV macaque model for primate lentiviral diseases.     

Patient-Specific iPSC-Derived Neural Differentiated and Hepatocyte-like Cells,Carrying the Compound Heterozygous Mutation p.V1023Sfs*15/p.G992R,Present the “Variant” Biochemical Phenotype of Niemann-Pick Type C1 Disease

Niemann–Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder caused by autosomal recessive mutations in the NPC1 gene. Patients display a wide spectrum on the clinical as well as on the molecular level, wherein a so-called “variant” biochemical phenotype can be observed. Here, we report an in vitro analysis of fibroblasts obtained from an NP-C1 patient carrying the undescribed compound heterozygous mutation p.V1023Sfs*15/p.G992R. Since NP-C1 is a neurovisceral disease and the patient suffers from severe neurological as well as hepatic symptoms, we extended our study to neural differentiated and hepatocyte-like cells derived from patient-specific induced pluripotent stem cells. We detected slightly increased intracellular cholesterol levels compared to the control cell line in fibroblasts, neural differentiated and hepatocyte-like cells, suggesting a “variant” biochemical phenotype. Furthermore, the total NPC1 protein, as well as post-ER glycoforms of the NPC1 protein, tended to be reduced. In addition, colocalization analysis revealed a mild reduction of the NPC1 protein in the lysosomes. The patient was diagnosed with NP-C1 at the age of 34 years, after an initial misdiagnosis of schizophrenia. After years of mild and unspecific symptoms, such as difficulties in coordination and concentration, symptoms progressed and the patient finally presented with ataxia, dysarthria, dysphagia, vertical supranuclear gaze palsy, and hepatosplenomegaly. Genetic testing finally pointed towards an NP-C1 diagnosis, revealing the so-far undescribed compound heterozygous mutation p.V1023Sfs*15/p.G992R in the NPC1 gene. In light of these findings, this case provides support for the p.G992R mutation being causative for a “variant” biochemical phenotype leading to an adult-onset type of NP-C1 disease.