Human whey was subjected to gel chromatography. Two folate binding protein fractions wer eluted: a major one (Mr congruent to 30,000) and a minor one (Mr greater than 200,000). The former folate binding fraction (FB) could be isolated from whey by means of ion exchange chromatography. Equilibrium dialysis experiments with whey and isolated FB indicated the existence of cooperatively interacting sites in folate binding. The folate analogue methotrexate inhibited folate binding. Inhibition was apparently of a competitive type since methotrexate did not reduce maximum binding capacity. Cooperativity was lost in the presence of methotrexate. Binding affinity was inversely proportional to the concentration of FB suggesting involvement of a polymerizing protein system in binding. None of the other fractions eluted on the ion exchange column could bind folate.

Glutamine:fructose-6-phosphate aminotransferases (GFATs) and phosphofructokinase (PFKs) are the principal rate-limiting enzymes involved in hexosamine biosynthesis pathway (HBP) and glycolysis pathway, respectively. In this study, the NlGFAT and NlPFK were knocked down through RNA interference (RNAi) in Nilaparvata lugens, the notorious brown planthopper (BPH), and the changes in energy metabolism were determined. Knockdown of either NlGFAT or NlPFK substantially reduced gene expression related to trehalose, glucose, and glycogen metabolism pathways. Moreover, trehalose content rose significantly at 72 h after dsGFAT injection, and glycogen content increased significantly at 48 h after injection. Glucose content remained unchanged throughout the experiment. Conversely, dsPFK injection did not significantly alter trehalose, but caused an extreme increase in glucose and glycogen content at 72 h after injection. The Knockdown of NlGFAT or NlPFK significantly downregulated the genes in the glycolytic pathway, as well as caused a considerable and significant decrease in pyruvate kinase (PK) activity after 48 h and 72 h of inhibition. After dsGFAT injection, most of genes in TCA cycle pathway were upregulated, but after dsNlPFK injection, they were downregulated. Correspondingly, ATP content substantially increased at 48 h after NlGFAT knockdown but decreased to an extreme extent by 72 h. In contrast, ATP content decreased significantly after NlPFK was knocked down and returned. The results have suggested the knockdown of either NlGFAT or NlPFK resulted in metabolism disorders in BPHs, highlighting the difference in the impact of those two enzyme genes on energy metabolism. Given their influence on BPHs energy metabolism, developing enzyme inhibitors or activators may provide a biological control for BPHs.

Melanocortin peptides have been reported to influence opiate tolerance, but the neuronal basis underlying these actions is unknown. We studied the contribution of melanocortin (MC4R) receptors to morphine effects. The MC4R mRNA level in the amygdala was decreased after acute morphine treatment and increased in rats tolerant to morphine as evidenced by quantitative real-time PCR method. Moreover, the intra-amygdalar microinjection of antagonist of MC4R attenuated morphine tolerance. Expression of the spinal MC4R after sciatic nerve injury was decreased in the early phase of neuropathy and slightly decreased 2-3 weeks after injury. These findings suggest that the altered melanocortin receptor function may contribute to the development of morphine-induced effects. Thus, the melanocortin receptors may be a target for development of better and more effective drugs for the therapy of chronic pain. Acknowledgement: Supported by KBN grant for the statutory activity. PS7-02Mouse and rat VRL-1 are both expressed in the dorsal root ganglion derived cell line F-11 L. Münter, R. Jahnel, M. Dreger, C. Gillen and F. Hucho Institute f ü r Chemie-Biochemie, FU Berlin, Thielallee 63, 14195 Berlin; Grü nenthal GmbH, Postfach 50 04 44, 52088, Aachen, Germany, lisamuen@chemie.fu-berlin.deThe TRP-channel VRL-1 is a heat-sensitive, nonselective cation channel expressed by primary sensory neurons and non-neuronal tissues. VRL-1 is activated above 52°C but not by capsaicin or protons. It has been shown that F-11 cells, hybridoma of mouse neuroblastoma and rat dorsal root ganglion cells, endogenously express VRL-1 (1). We here present data on the characterisation of VRL-1 in F-11 cells at the nucleic acid and protein level. Anti-VRL-1 immunoreactivity showed up at 80 kDa in Western blots with F-11 cell homogenate. By analysing overlapping PCRfragments with specific rat VRL-1 primers we found that F-11 cells express both rat and mouse VRL-1. The F-11 cell line provides a powerful experimental system to study VRL-1 biochemistry, but one has to be aware that the mouse and the rat protein are expressed. Reference PS7-03Expression of the N-and C-termini from the Vanilloid Receptor 1 (VR1) as MBP-fusion proteins for affinity purification and search for interaction partners R. Jahnel, C. Goswami, H. Si, M. Dreger, C. Gillen and F. Hucho Institute f ür Chemie-Biochemie, FU Berlin, Thielallee 63, 14195 Berlin; Grü nenthal GmbH, Postfach 50 04 44, 52088 Aachen, Germany, rjahnel@chemie.fu-berlin.deThe Vanilloid Receptor 1 is a heat-sensitive, nonselective cation channel expressed by primary sensory neurons involved in nociception. To find and study potential interacting proteins of VR1 we cloned the full N-and C-termini without transmembrane sequences of VR1 into the bacterial expression vector pMALc2x. We here report the expression and purification of the VR1 fragments fused to the maltose binding protein (MBP). In addition, we assessed the subcellular targeting of N-and C-terminal VR1 fragments expressed in the cell line F-11. This cell line has...