Production of human UDP-glucuronosyltransferases 1A6 and 1A9 using the Semliki Forest virus expression system.

Human UDP-glucuronosyltransferases (UGTs) 1A6 and 1A9 were expressed using Semliki Forest virus (SFV) vectors. Infection of chinese hamster lung fibroblast V79 cells with recombinant SFV-UGT viruses resulted in efficient protein expression as detected by metabolic labeling, Western blot analyses and immunofluorescence microscopy. The expression of UGT 1A6 and UGT1A9 in the SFV-infected cells was approximately two fold higher than in a stable V79 cell line. No UGT signal was detected in noninfected cells. In addition, SFV-UGT viruses also efficiently infected other mammalian cells, such as baby hamster kidney (BHK), chinese hamster ovary (CHO) and human lung (WI-26 VA4) cells leading to high production of recombinant enzyme. The measurement of enzyme activities and kinetic parameters using p-nitrophenol and nitrocatechol (entacapone) as substrates for UGT1A6 and UGT1A9, respectively, showed that the overall kinetic properties of the enzymes produced by the two systems were similar. We conclude that the SFV expression system represents an efficient, fast and versatile method for production of metabolic enzymes for in vitro assays.

Effect of the umami peptides on the ligand binding and function of rat mGlu4a receptor might implicate this receptor in the monosodium glutamate taste transduction.

1. The effect of several metabotropic ligands and di- or tripeptides were tested on the binding of [3H]-L(+)-2-amino-4-phosphonobutyric acid ([3H]-L-AP4) on rat mGlu4 receptor. For selected compounds, the functional activity was determined on this receptor using the guanosine-5'[gamma-35S]-thiotriphosphate [gamma-35S]-GTP binding assay. 2. Using the scintillation proximity assay, [3H]-L-AP4 saturation analysis gave binding parameters K(D) and Bmax values of 150 nM and 9.3 pmoles mg-1 protein, respectively. The specific binding was inhibited concentration-dependently by several mGlu receptor ligands, and their rank order of affinity was established. 3. Several peptides inhibited the [3H]-L-AP4 binding with the following rank order of potency: glutamate-glutamate>glutamate-glutamate-leucine=aspartate - glutamate>>glutamate - glutamate-aspartate>lactoyl-glutamate>>aspartate-aspartate. Aspartate-phenylalanine-methyl ester (aspartame) was inactive up to 1 mM and guanosine-5'-monophosphate and inosine-5'-monophosphate were inactive up to 100 micronM. 4. The [gamma-35S]-GTP binding functional assay was used to determine the agonist activities of the different compounds. For the rat mGlu4 agonists, L-AP4 and L-glutamate, the correlation between their occupancy and activation of the receptor was close to one. The peptides, Glu-Glu, Asp-Glu and Glu-Glu-Asp failed to stimulate the [gamma-35S]-GTP binding at receptor occupancy greater than 80% and Glu-Glu-Leu appeared to be a weak partial agonist. These peptides did not elicit a clear dose-dependent umami perception. However, Glu-lac showed a good correlation between its potency to stimulate the [gamma-35S]-GTP binding and its affinity for displacement of [3H]-L-AP4 binding. These data are in agreement with the peptide taste assessment in human subjects, which showed that the acid derivatives of glutamate had characteristics similar to umami.

Expression and intracellular localisation of odorant receptors in mammalian cell lines using Semliki Forest virus vectors.

Odorant receptors are members of the G protein-coupled receptor superfamily. They are expressed on the surface of cilia of olfactory neurons, where they bind ligand (odorant). Studies of the molecular mechanisms of olfaction are complicated by the extremely large number of receptor genes, and difficulties in pairing a particular mammalian receptor to a specific odorant ligand in vivo. Here we report expression and localisation studies of two rat odorant receptor genes (17 and OR5), and C. elegans odr-10, using the Semliki Forest virus (SFV) system. All receptors were epitope-tagged at the N- or C-terminus in order to facilitate their detection in infected cells, and determine the localisation and membrane-orientation of recombinant proteins. The immortalised mouse olfactory neuronal cell line OLF 442, rat cortical and striatal primary neuron cultures, and the baby hamster kidney (BHK) cells, were infected and tested. Immunofluorescence and confocal microscopy studies performed on permeabilised, non-permeabilised and native cells revealed that in BHK cells the rat receptors 17 and OR5 were not targeted to the plasma membrane and remained in the endoplasmic reticulum. In contrast, in the mouse olfactory cell line OLF 442 both rat receptors were correctly inserted into the plasma membrane. Similar results were obtained using primary neurons, indicating that like mature neurons, the immortalised OLF 442 cells are capable of providing for correct odorant receptor processing and targeting.

[The effect of the morphofunctional state of the thrombocytes on the activation of destabilization processes in patients with acute forms of ischemic heart disease]. / Vliianie morfofunktsional'nogo sostoianiia trombotsitov na aktivatsiiu protsessov destabilizatsii u bol'nykh s ostrymi formami ishemicheskoi bolezni serdtsa.

With the purpose of assessing the influence that platelet morphofunctional state may have on the degree of destabilization in patients with different forms of unstable angina we examined 198 patients using stress tests, Holter ECG monitoring, studying activation of thrombocytes with the aid of a luminescent-microscopy technique, and performing an analysis of aggregation. Important differences have been revealed in the studied parameters in those patients presenting with freshly detected and progressive stenocardia and with a developing, despite of the treatments administered, myocardial infarction, that might be caused by abnormal shifts in the thrombocyte intact/activated forms ratio.