Presenilin-1 is processed into two major cleavage products in neuronal cell lines.

Presenilin-1 (PS-1) has been identified as the protein encoded by the chromosome 14 locus that, when mutated, leads to familial Alzheimer's disease (FAD). Using PS-1 transfected SHSY5Y neuroblastoma cells, we have demonstrated by immunodetection, using polyclonal antibodies, that PS-1 is processed to give two fragments: an N-terminal 28 kDa fragment, and a C-terminal 18 kDa fragment. In a number of non-transfected cell types, most PS-1 is detected as the cleaved products. The molecular weights of the PS-1 cleavage products suggest that the cleavage point will most probably be within a region of the hydrophilic loop domain coded for by either exon 8 or 9 of the PS-1 gene. The clustering of FAD mutations within exon 8 strongly suggests that it encodes a key functional domain. It seems likely that the cleavage of PS-1 is crucial to some aspect of its functionality. An understanding of this process will give insights into the pathology of AD, and may offer new opportunities for therapeutic intervention.

Expression of hybrid malaria antigens in insect cells and their engineering for correct folding and secretion.

Hybrid proteins containing selected regions of the major surface antigens of the sporozoite and merozoite stages of Plasmodium falciparum were expressed in insect cells using baculovirus vectors. A recombinant protein containing the signal peptide from the precursor to the major merozoite surface antigens (PMMSA) fused to a fragment from the carboxy (C) terminus of the same gene was recognized by monoclonal antibodies specific for reduction-sensitive conformational epitopes within the C-terminal fragment, suggesting that correct disulphide cross-linking of cysteine residues within this region had occurred. Addition of 26 copies of the tetrapeptide repeat from the circumsporozoite protein (CSP) resulted in a protein recognized by anti-CSP antiserum as well as the conformation specific monoclonal antibodies. Deletion of the C-terminal putative anchor sequence from both proteins resulted in secretion of protein in a fully soluble form antigenically indistinguishable from the anchor containing products. Correct conformation was not observed when the proteins were expressed as polyhedrin fusions without the signal peptide. These data indicate that the PMMSA signal peptide is recognized in insect cells and that correct assembly of disulphide cross-links is dependent upon targeting the protein to the endoplasmic reticulum.

Expression of foreign genes in cultured insect cells using a recombinant baculovirus vector.

An important consideration for the expression of cloned genes in recombinant expression systems is the ability of the foreign host to produce the protein faithfully in a form that is similar or identical to that found in the cell type from which the gene was cloned. For eukaryotic proteins, this frequently involves many posttranslational modifications of the protein, such as glycosylation, phosphorylation, processing, and secretory events. Additionally, very precise interactions are essential for the correct folding of the polypeptide to achieve the final tertiary structure. If the folding is incorrect, then the molecule will often be biologically inactive.

The action of GABA receptor agonists and antagonists on muscle membrane conductance in Schistocerca gregaria.

1. The properties of postsynaptic gamma-aminobutyric acid (GABA) receptors in the extensor tibiae muscle of Schistocerca gregaria were studied by conventional electrophysiological recording techniques. 2. GABA and other active GABA receptor agonists produced rapid, dose-dependent, reversible increases in membrane conductance. 3. In two microelectrode experiments the ED50 for GABA was approximately 1 mM. In three microelectrode experiments (assuming short cable theory conditions) the ED50 for GABA was 2.3 mM. The Hill coefficient for GABA estimated from the latter experiments was 1.4. 4. The relative potency of muscimol/GABA at the ED50 for GABA was 1.36. 3-Aminopropane sulphonic acid (3-APS) and isonipecotic acid were weakly active, baclofen and piperidine-4-sulphonic acid (P4S) were inactive. Isoguvacine produced depolarizations and increases in conductance in preparations which hyperpolarized in response to GABA. These depolarizations were enhanced by both picrotoxin and pitrazepin although the increases in input conductance were depressed. 5. Picrotoxin (20 microM), (+)-bicuculline (20-100 microM) and pitrazepin (1-10 microM) all reversibly antagonized GABA-induced responses. Such antagonism was not competitive in the case of picrotoxin and (+)-bicuculline but was competitive for pitrazepin. Schild plot analysis gave an average pA2 value of 5.5 for pitrazepin. 6. The significance of these results is briefly discussed.