Leo A. Ginsel

Congenital sucrase-isomaltase deficiency. Identification of a glutamine to proline substitution that leads to a transport block of sucrase-isomaltase in a pre-Golgi compartment.

Congenital sucrase-isomaltase deficiency is an example of a disease in which mutant phenotypes generate transport-incompetent molecules. Here, we analyze at the molecular level a phenotype of congenital sucrase-isomaltase deficiency in which sucrase-isomaltase (SI) is not transported to the brush border membrane but accumulates as a mannose-rich precursor in the endoplasmic reticulum (ER), ER-Golgi intermediate compartment, and the cis-Golgi, where it is finally degraded. A 6-kb clone containing the full-length cDNA encoding SI was isolated from the patients intestinal tissue and from normal controls. Sequencing of the cDNA revealed a single mutation, A/C at nucleotide 3298 in the coding region of the sucrase subunit of the enzyme complex. The mutation leads to a substitution of the glutamine residue by a proline at amino acid 1098 (Q1098P). The Q1098P mutation lies in a region that is highly conserved between sucrase and isomaltase from different species and several other structurally and functionally related proteins. This is the first report that characterizes a point mutation in the SI gene that is responsible for the transport incompetence of SI and for its retention between the ER and the Golgi.

Visualizing drug transport across stratum corneum: cryotechniques, vapour fixation, autoradiography

Abstract The potentials of a method for visualizing the transport of drugs across human stratum corneum at electron microscopic level were investigated. Possible extraction and/or dislocation of the drug during sample preparation for electron microscopy was reduced as much as possible. In this “dry” sample preparation drug-loaded human skin was rapidly frozen, freeze-dried, osmium tetroxide vapour fixed, and embedded in Epon under vacuum. Visualization of the drug was achieved by using electron microscope autoradiography (general applicability). The model compound in the autoradiography studies was tritiated hydrocortisone. No detectable extraction of drug was measured during the “dry” sample preparation. Furthermore, this method preserved the ultrastructure of stratum corneum very well. The combination of the “dry” sample preparation and electron microscope autoradiography was feasible and there were no problems regarding the detection limit; the only remaining problem was an unsatisfactory resolution. However, considering the dimensions of the inter- and intracellular domains and the resolution attainable for electron microscope autoradiography, it should be possible to discriminate between intercellular and transcellular drug transport across stratum corneum.

Sorting of Endogenous Proteins in Intestinal Epithelial Cells

The intestinal epithelial cell (enterocyte) forms an interesting type of polarized cell to study the sorting of endogenous membrane proteins (for reviews see Ginsel et al., 1988; Fransen et al., 1988; and Hauri, 1988). It is characterized by a typical brush border (apical or microvillar) membrane containing hydrolases involved in the terminal digestion of dietary carbohydrates and peptides, and a basolateral membrane with different structural and functional properties. Studies on the pathways and mechanisms of protein sorting in this type of cell have been going on for a relatively long period. The data on the transport pathways were however, controversial. It was unclear whether the brush-border hydrolases are directly transported from the Golgi apparatus to the apical membrane (Danielsen and Cowell, 1985; Fransen et al., 1985) or, indirectly, via the basolateral membrane (Hauri et al., 1979; Massey et al., 1987).