The projections from the anterior (NIA) and posterior (NIP) interposed nuclei to the magnocellular red nucleus (RNm) have been investigated in the rat, using the retrograde transport of horseradish peroxidase-wheatgerm agglutinin conjugate. Projections from the NIA extend throughout the RNm, whereas those from the NIP only reach its medial aspect. In addition, a topographical organization of the NIA-RNm pathway was found, such that the medial NIA projects ventrally, the lateral NIA projects dorsally.

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PubMedDate not supplied

The red nucleus activity in rats deprived of the inferior olivary complex.

The long-term effects of the inferior olive destruction on the red nucleus activity, were studied in the rat following injection of 3-acetylpyridine. As soon as the olivary activity was suppressed, the discharge of the rubral units drastically decreased. Then, they progressively recovered the control frequency during the first month, although a normal rubral activity was not restored up to 8 months. The hypothesis is advanced that the olivocerebellar system is essential to shape the activity of the rubrospinal pathway.

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PubMedDate not supplied

[Brief history of long-term synaptic depression of the cerebellum].

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Expression of the mammalian renal peptide transporter PEPT2 in the yeast Pichia pastoris and applications of the yeast system for functional analysis.

It has recently been identified the PEPT2 cDNA encodes the high affinity proton-coupled peptide transporter in rabbit kidney cortex. PEPT2 represents a 729 amino acid protein with 12 putative transmembrane domains that mediates H+/H3O+ dependent electrogenic transmembrane transport of di- and tripeptides and of selected peptidomimetics. Here the functional expression of PEPT2 in the methylotropic yeast Pichia pastoris is described under the control of a methanol inducible promoter. Western blot analysis of Pichia cell membranes prepared from a recombinant clone identified a protein with an apparent molecular mass of about 85-87 kDa. Peptide uptake into cells expressing PEPT2 was up to 80 times higher than in control cells. Cells of recombinant clones showed a saturable peptide transport activity for the hydrolysis resistant dipeptide 3H-D-Phe-Ala with an app. K0.5 of 0.143 +/- 0.016 mM. Inhibition of 3H-D-Phe-Ala uptake by selected di- and tripeptides and beta-lactam antibiotics revealed the same substrate specificity as obtained in renal membrane vesicles or for PEPT2 when expressed in Xenopus laevis oocytes. A novel fluorescence based assay for assessing transport function based on a coumarin-labeled fluorescent peptide analogue has also been developed. Moreover, using a histidyl auxotrophe strain a PEPT2 expressing cell clone in which transport function can be monitored by a simple yeast growth test was established. In conclusion, this is one of only a few reports on successful functional expression of mammalian membrane transport proteins in yeast. The high expression level will provide a simple means for future studies either on the structure-affinity relationship for substrate interaction with PEPT2 or for selection of mutants generated by random mutagenesis.

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