Expression cloning human and rat renal cortex Na/Pi cotransporters: behind the scenes in the Murer laboratory.

In the pre-genomic era, the cloning of a cDNA represented a significant achievement, particularly if the gene of interest encoded a membrane protein. At the time, molecular probes such as partial peptide sequences, suitable nucleic acid sequences, or antibodies were unavailable for most proteins and the "sodium-phosphate transporter" was no exception. In contrast, brush-border membrane vesicles and epithelial cell culture experiments had established a reliable set of functional hallmarks that described Na-dependent phosphate transport activity in some detail. Moreover, aspects of hormonal regulation of phosphate homeostasis could be recapitulated in these model systems. Expression cloning elegantly combined functional protein expression in Xenopus laevis oocytes with molecular biology to overcome the lack of molecular probes.

Functional expression of type 1 rat GABA transporter in microinjected Xenopus laevis oocytes.

In this chapter we describe technical aspects and experimental potential of the two electrodes voltage clamp (TEVC) electrophysiological approach applied to the Xenopus oocyte-expression system. This technique is addressed to the study of a particular class of expressed proteins, those responsible to drive ion fluxes through the plasma membrane. In fact the voltage-clamp technique provides the most direct and sensitive measurement of the functional properties of ion channels and electrogenic transporters, allowing specific ion currents to be recorded under well-defined voltage conditions and temporal control. Besides the study of the physiological properties of specific ion channels as well as their pharmacological modulation, further applications of the TEVC on oocytes include the possibility to introduce single point mutations in the channel construct and to infer to possible structural aspects and functional involvements of single amino acidic residues. To achieve these results these technique should be strictly tied to basic molecular biology techniques. Recent advance of this technique in drug discovery procedures have been briefly enlightened.

Glial and neuronal alterations in the nucleus tractus solitarii of sudden infant death syndrome victims.

The factors underlying the sudden infant death syndrome (SIDS) are still unknown, but in recent years much attention has been focused on the central cardiorespiratory control system. In the present work we analyzed the nucleus tractus solitarii (nTS) of 23 SIDS victims and 17 age-matched control cases. We studied the functional and morphological alterations of neurons and glial cells to evaluate the results of possible hypoxic-ischemic injury that could have led to sudden death. Morphometric and immunohistochemical analyses were performed on medullary sections. In the nTS of SIDS victims we observed modifications of both neuronal and glial cells. Brain injury triggers the activation of both astrocytes and microglia, which respond to neuronal damage by characteristic changes that could explain our observations in the nTS of SIDS victims. In our investigation of the nTS of SIDS victims we found a significant increase of reactive astrocytes density, a significantly higher percentage of necrotic cells, an increase of reactive microglial cells density, a significantly higher expression of substance P and the presence of NMDA receptors immunoreactivity. Our results support the hypothesis that there is injury of the nTS neurons in SIDS victims, even if the causes of this damage are still unknown. This neuronal damage may explain why adequate ventilation is often not maintained during hypoxia. Such histological findings have never been thought sufficient to explain SIDS, but the tissue findings could be an indication of the impairment of several pathophysiological mechanisms which may underlie brainstem dysfunction, affecting cardiorespiratory control.