Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed.

For years, an increasing number and diversity of genetically modified plants has been grown on a commercial scale. The need for detection and identification of these genetically modified organisms (GMOs) calls for broad and at the same time flexible high throughput testing methods. Here we describe the development and validation of a hexaplex real-time polymerase chain reaction (PCR) screening assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay. The assay comprises detection systems for Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens NOS terminator, Figwort Mosaic Virus 34S promoter and two construct-specific sequences present in novel genetically modified soybean and maize that lack common screening elements. Additionally a detection system for an internal positive control (IPC) indicating the presence or absence of PCR inhibiting substances was included. The six real-time PCR systems were allocated to five detection channels showing no significant crosstalk between the detection channels. As part of an extensive validation, a limit of detection (LOD(abs)) < or = ten target copies was proven in hexaplex format. A sensitivity < or = ten target copies of each GMO detection system was still shown in highly asymmetric target situations in the presence of 1,000 copies of all other GMO targets of each detection channel. Furthermore, the applicability to a broad sample spectrum and reliable indication of inhibition by the IPC system was demonstrated. The presented hexaplex assay offers sensitive and reliable detection of GMOs in processed and unprocessed food, feed and seed samples with high efficiency.

Succinylcholine metabolite succinic acid alters steady state activation in muscle sodium channels.

BACKGROUND: Animal experiments revealed that succinylcholine produced masseter muscle rigidity and activated myotonic discharges despite neuromuscular blockade with a nondepolarizing blocker. These results suggest that either succinylcholine or its metabolites might interfere directly with voltage-operated ion channels of the sarcolemma. The aim of this study was to examine effects of one product of succinylcholine hydrolysis, succinic acid, on voltage-gated muscle sodium (Na+) channels. METHODS: Alpha subunits of human muscle sodium channels were heterologously expressed in HEK293 cells. Activation of Na+ currents was examined applying standard whole-cell voltage-clamp protocols in the absence (control and washout) and presence of succinic acid in different concentrations (0.05-10 mm). RESULTS: Succinic acid shifted the midpoints of steady state activation plots in the direction of more negative test potentials, indicating that channels open during smaller depolarizations in the presence of the drug. The maximum amount of the negative shift in 10 mm succinic acid was -6.3 +/- 1.7 mV; the EC50 for this effect was 0.39 mm. In addition, succinic acid (10 mm) significantly enhanced maximum currents after depolarizations with respect to a series of control experiments. CONCLUSION: Succinic acid facilitates voltage-dependent activation in muscle sodium channels in vitro. This might lead to muscle hyperexcitability in vivo.

Different effects of mexiletine on two mutant sodium channels causing paramyotonia congenita and hyperkalemic periodic paralysis.

Effects of the antiarrhythmic and antimyotonic drug mexiletine were studied on two sodium channel mutants causing paramyotonia congenita (R1448H) and an overlap paramyotonic and hyperkalemic paralytic syndrome (M1360V). Channels were expressed in human embryonic kidney cells and studied electrophysiologically, using the whole-cell patch-clamp technique. Compared to the wild-type, channel, both mutants showed alterations of inactivation, i.e. slower inactivation, left shift of steady-state inactivation and faster recovery from inactivation. Mexiletine caused a significantly larger use-dependent block of the R1448H mutant when compared to M1360V and wild-type channels. This can be explained by a prolonged recovery from mexiletine block as observed for R1448H channels, since the affinity of mexiletine for the inactivated state was similar for all three clones. The use-dependent block of sodium channels by mexiletine reduces repetitive series of action potentials and therefore improves muscle stiffness in myotonic patients. The enhanced use-dependent block as seen with R1448H may explain the extraordinary therapeutic efficacy of mexiletine in most patients with paramyotonia congenita.

The small sodium-channel blocking factor in the cerebrospinal fluid of multiple sclerosis patients is probably an oligopeptide.

An endogenous factor that is able to reduce the fast transient sodium current of excitable cells has been reported to exist in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. This was confirmed with nine clinically definite MS patients in the acute relapse. In order to purify and chemically identify the factor, microconcentration and gel filtration high-performance liquid chromatography (HPLC) were applied. After each purification step the activity-containing fraction was determined using a biological assay. With all CSFs the activity was contained in the fraction corresponding to 600-800 Da molecular weight, indicating that the factor is chemically homogeneous. The biological activity of the CSF specimens was not correlated to the laboratory CSF data; however, it was correlated to the area under the 210 nm UV light absorption peak in the corresponding chromatogram, i.e. the 600-800 Da MW fraction. As the factor was degradable by acid hydrolysis and a carboxypeptidase, it is suggested that it might be a small peptide.

Voltage-dependent block of normal and mutant muscle sodium channels by 4-Chloro-m-Cresol.

1 The effects of 4-Chloro-m-Cresol (4-CmC) were examined on heterologously expressed wild type (WT), Paramyotonia Congenita (R1448H) and Hyperkalemic Periodic Paralysis (M1360V) mutant alpha-subunits of human muscle sodium channels. 2 Block of rested sodium channels caused by 4-CmC was concentration-dependent with an ECR50 of 0.40 mM in WT, 0.45 mM in R1448H and 0.49 mM in M1360V. 3 Inactivation significantly promoted 4-CmC-induced sodium channel block in all clones indicated by 4-CmC-induced shifts of steady-state availability curves, reflecting a higher proportion of channel block at depolarized membrane potentials. Channel block was almost complete (>90%) at concentrations close to the ECR50 (0.5 mM) on application of an inactivating prepulse before the test pulse. 4 4-CmC accelerated the current decay following depolarization and prolonged recovery from inactivation in all clones. Of these, R1448H, the mutant which displayed severely impaired inactivation in the controls, responded to 4-CmC with the most pronounced acceleration of inactivation. Control experiments revealed enhanced recovery from inactivation in the mutants, which was restored to normal in 0.1 mM 4-CmC. 5 4-CmC induced no additional frequency-dependent block. 6 Our results clearly demonstrate that 4-CmC is as effective as lidocaine (Fan et al., 1996) in blocking muscle sodium channels. Low concentrations of the compound (

Structure of genes encoding chromosomal HMG1 proteins from maize.

The high mobility group (HMG) proteins of the HMG1 family are architectural proteins in chromatin that are considered to facilitate the formation of complex nucleoprotein structures in various biological processes such as transcription and recombination. Plants express a variety of these non-sequence-specific DNA-bending proteins. The sequences encoding the maize HMGa and HMGc1 proteins were isolated from a genomic DNA library. Determination of the nucleotide sequences of these genes revealed that the coding region of both genes has a similar genomic structure, comprising seven exons and six introns. The positioning of the introns is conserved between the two genes, whereas the number of introns and their positions are entirely different in the related animal genes. In the 5' flanking region of the hmgc1 gene, a copia-like retrotransposon was identified. In addition to the genes encoding HMGa and HMGc1, several genomic fragments (retropseudo gene, fragments of the genes) were isolated and characterised.

Cerebrospinal fluid and serum from patients with inflammatory polyradiculoneuropathy have opposite effects on sodium channels.

The effects of cerebrospinal fluid (CSF) and serum from patients having Guillain-Barré syndrome (GBS) or chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) on voltage-dependent Na+ channels were compared. Bathing human myoballs in CSF substantially reduced their Na+ currents (by > 40% with 8 of 10 patients) elicited at 1 Hz under whole-cell recording conditions. This was because, at the resting potential, more Na+ channels were inactivated (left-shift of the h infinity curve). CSF from patients with other neurological diseases (OND) produces a similar, but smaller, effect. In contrast, serum samples from the same GBS and OND patients caused an increase of the Na+ currents by reducing the number of Na+ channels inactivated at the resting potential. This right-shift of the h infinity curve is in part explained by the effect of serum albumin. We confirm that the CSF of most GBS and CIDP patients contains factors inhibiting voltage-dependent Na+ currents. There is no indication that such factors are effective in the serum of these patients.

Isolation and characterization of high-mobility-group proteins from maize.

Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5' flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.

Age-related determination of the basal and calmodulin-stimulated human RBC Ca2(+)-ATPase activity.

The basal and calmodulin-stimulated activity of the human erythrocyte Ca2(+)-ATPase was determined in 56 healthy individuals of different ages in order to set control values. The basal activity of all 56 healthy subjects was 0.805 +/- 0.525 mumole hydrolyzed ATP/mg protein/hr, while the calmodulin-stimulated activity gave an average of 2.437 +/- 0.785 mumole hydrolyzed ATP/mg protein/hr. There was no significant difference in basal Ca2(+)-ATPase activity in males and females; however, significantly increased levels of the stimulated red cell calcium pump was seen in females. Based on age, the basal activity for newborns and children up the age of 7 years was higher than that for adults and a distinct increase in activation by calmodulin was observed with a maximum at the age range of 8-13 years. It was demonstrated that for the investigation of Ca2(+)-ATPase activities in children different normal values must be taken into consideration than for those already reported for adults. Our data may serve as controls for Ca2(+)-ATPase activities for comparison purposes and for further studies from different diseases in childhood where increased intraerythrocytic calcium levels are reported.