[Rapid determination of residues of 4 tetracyclines in meat by a microbiological screening, HPLC and LC/MS/MS].

A rapid and precise determination residues of 4 tetracyclines (TCs) (oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DOXY)) in meat was developed by employing three analyses; a microbiological screening, HPLC and LC/MS/MS. TCs were extracted with pH 4.0 McIlvaine buffer containing 0.01 mol/L EDTA from a meat sample, and then purified using a mixed mode, reversed-phase and cation-exchange cartridge. The mean recoveries (n=5) of 0.2 microg/g OTC, TC and CTC, 0.05 microg/g DOXY spiked in meat samples were 76.6-99.0% (C.V. 1.6-5.4%). In 13 meat samples in which the microbiological screening indicated the presence of TCs, CTC (9 samples) and DOXY (4 samples) were identified with HPLC and LC/MS/MS.

Complementary DNA cloning and characterization of RANDAM-2, a type I membrane molecule specifically expressed on glutamatergic neuronal cells in the mouse cerebrum.

A membrane-surface glycoprotein, RANDAM-2, is one of the neuronal cell lineage-specific antigens involved in the neuronal differentiation of P19 embryonic carcinoma (EC) cells and the mouse central nervous system (CNS). Complementary DNA cloning of RANDAM-2 indicated that its nucleotide sequence completely matched that of PA2.26 antigen, a sialomucin-like transmembrane glycoprotein previously found on tumorigenic keratinocytes. RANDAM-2 transcripts were detectable from the embryonic stage of 6.5 days, and then the expression continued throughout the remaining embryonic stages and adulthood, with a localization restricted to the CNS. In growth factor-induced neurospheres and adult cerebrum, RANDAM-2-expressing cells coincided well not only with nestin-positive cells but also with glutamate-positive neurons, but not with gamma-aminobutyric acid-positive ones. These results indicate that RANDAM-2 is one of the type I membrane surface antigens constitutively expressed on undifferentiated neuronal cells and the glutamatergic neuronal cells during mouse neurogenesis.

Immunohistochemical and biochemical analyses of GD3, GT1b, and GQ1b gangliosides during neural differentiation of P19 EC cells.

In an earlier study, we showed that expressions of GD3, GT1b, and GQ1b gangliosides in P19 embryonic carcinoma (EC) cells were enhanced during their neural differentiation induced by retinoic acid. We now further demonstrated that this increase of the b-series gangliosides is due to an increase in their corresponding synthases (sialyltransferase-II, -IV, and -V) in the Golgi. Of the three gangliosides studied, GQ1b appeared to be the best candidate for monitoring such differentiation process. We also used fluorescence-labeled monoclonal antibodies and confocal fluorescence microscopy to obtain direct visual information about the relationship of gangliosides and neural specific proteins in neuron development. Again, GQ1b is the most interesting as it localizes with synaptophysin and neural cell adhesion molecules (NCAMs) on synaptic boutons or dendritic spines in RA-induced neurons (R/N). This suggests that GQ1b could be used as a marker for synapse formation during construction of the neural network.

Identification of neuronal cell lineage-specific molecules in the neuronal differentiation of P19 EC cells and mouse central nervous system.

P19 embryonic carcinoma (EC) cells are one of the simplest systems for analyzing the neuronal differentiation. To identify the membrane-associated molecules on the neuronal cells involved in the early neuronal differentiation in mice, we generated two monoclonal antibodies, SKY-1 and SKY-2, by immunizing rats with a membrane fraction of the neuronally committed P19 EC cells as an antigen. SKY-1 and SKY-2 recognized the carbohydrate moiety of a 90 kDa protein (RANDAM-1) and the polypeptide core of a 40 kDa protein (RANDAM-2), respectively. In the P19 EC cells, the expression of RANDAM-1 was colocalized to a part of Nestin-positive cells, whereas that of RANDAM-2 was observed in most Nestin-positive cells as well as beta-III-tubulin positive neurons. In the embryonic and adult brain of mice, RANDAM-1 was expressed at embryonic day 8.5 (E8.5), and the localization of antigen was restricted on the neuroepithelium and choroid plexus. The RANDAM-2 expression commenced at E6.0, and the antigen was distributed not only on the neuroepithelium of embryonic brain but on the neurons of adult brain. Collectively, it was concluded that RANDAM-1 is a stage specific antigen to express on the neural stem cells, and RANDAM-2 is constitutively expressed on both the neural stem cells and differentiated neuronal cells in mouse central nervous system (CNS).

Pax6 controls the expression of Lewis x epitope in the embryonic forebrain by regulating alpha 1,3-fucosyltransferase IX expression.

Pax6 is a transcription factor involved in brain patterning and neurogenesis. Expression of Pax6 is specifically observed in the developing cerebral cortex, where Lewis x epitope that is thought to play important roles in cell interactions is colocalized. Here we examined whether Pax6 regulates localization of Lewis x using Pax6 mutant rat embryos. The Lewis x epitope disappeared in the Pax6 mutant cortex, and activity of alpha1,3-fucosyltransferase, which catalyzed the last step of Lewis x biosynthesis, drastically decreased in the mutant cortex as compared with the wild type. Furthermore, expression of a fucosyltransferase gene, FucT-IX, specifically decreased in the mutant, while no change was seen for expression of another fucosyltransferase gene, FucT-IV. These results strongly suggest that Pax6 controls Lewis x expression in the embryonic brain by regulating FucT-IX gene expression.