Lydia Hendriks

Flemish and Dutch Mutations in Amyloid β Precursor Protein Have Different Effects on Amyloid β Secretion

Mutations in the amyloid beta precursor protein (APP) gene cosegregate with autosomal dominant Alzheimer disease (AD). Brain pathology of AD is characterized by amyloid deposition in senile plaques and by neurofibrillary tangles. Amyloid deposits in AD brains consist of amyloid beta (A beta), a 4-kDa proteolytic product of APP. In contrast, two other mutations in APP, the Flemish APP692 and Dutch APP693 mutations, are associated with autosomal dominant cerebral hemorrhages due to congophilic amyloid angiopathy (CAA) in the presence or absence of AD pathology, respectively. Both mutations are located within A beta near the constitutive cleavage site. While a common effect of AD-linked mutations is to elevate A beta 42 extracellular concentrations, not much is known about the effect of APP692 and APP693. Here we provide evidence that APP692 and APP693 have a different effect on A beta secretion as determined by cDNA transfection experiments. While APP692 upregulates both A beta 40 and A beta 42 secretion, APP693 does not. These data corroborate with previous findings that increased A beta secretion and particularly of A beta 42, is specific for AD pathology.

Nonviral transfection of distinct types of human dendritic cells: high-efficiency gene transfer by electroporation into hematopoietic progenitor- but not monocyte-derived dendritic cells

Human dendritic cells (DC) are highly professional antigen presenting cells for the priming of naive cytotoxic T cells. Gene transfer in DC would be a useful strategy to load DC with relevant de novo synthesized antigens for immunotherapeutical purposes. As a first step towards a DC-based gene therapy, we examined the efficiency of nonviral transfection in different types of cultured human dendritic cells with a humanized red-shifted green fluorescent protein reporter gene. Plasmid DNA transfection by electroporation or lipofection was used to transfect CD34+ progenitor cell-derived DC (PC-DC) and Langerhans’ cells (PC-LC), as well as monocyte-derived DC (Mo-DC). While lipofection was unsuccessful in all types of DC, we obtained high-efficiency gene transfer by electroporation in PC-LC (16%) and PC-DC (12%). In contrast, electroporation was strikingly less efficient in Mo-DC (⩽2%). The potent allostimulatory capacity of DC was still retained in electroporated PC-DC and PC-LC. In conclusion, electroporation of antigen expressing plasmid DNA is an efficient tool for nonviral gene transfer in PC-DC and PC-LC, but not in Mo-DC and could be useful for the development of DC-based tumor immunotherapy.

The Nucleotide-sequence of the Small Ribosomal-subunit Rna of the Yeast Candida-albicans and the Evolutionary Position of the Fungi Among the Eukaryotes

Summary Up to now the small ribosomal subunit RNA sequences of about 50 different eukaryotes have been published, of which only three belong to the fungi. We determined the complete srRNA sequence of the imperfect yeast Candida albicans. The sequence is 1788 nucleotides long and was determined at the DNA level using the dideoxy method with a set of primers specific for conserved sequences of small ribosomal subunit RNA. An evolutionary tree, comprising 58 organisms including C. albicans, was constructed. This tree shows a number of early diverging lineages such as a diplomonad, a microsporidian, an amoeba, slime molds, an euglenoid, kinetoplastids and sporozoans. Next within a relatively short time interval there is a radiation into a number of clusters composed of ciliates, metazoa, fungi and green plants. C. albicans was previously classified in the artificial taxon of imperfect fungi. The evolutionary tree presented in this paper clearly shows C. albicans to belong to the ascomycetous yeasts. An additional aim of this study was the refinement of the srRNA secondary structure model. Although the outline of this model is now well established, no consensus model exists in certain eukaryote-specific areas of high structural variability. The srRNA sequence of xC. albicans was fitted into the secondary structure model and the existence of a pseudoknot is proposed in one of these eukaryote-specific areas.

Presenile Alzheimer dementia characterized by amyloid angiopathy and large amyloid core type senile plaques in the APP 692Ala→Gly mutation

Abstract Mutations at codons 717 and 670/671 in the amyloid precursor protein (APP) are rare genetic causes of familial Alzheimer’s disease (AD). A mutation at codon 693 of APP has also been described as the genetic defect in hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). We have reported a APP692Ala→Gly (Flemish) mutation as a cause of intracerebral hemorrhage and presenile dementia diagnosed as probable AD in a Dutch family. We now describe the post-mortem examination of two demented patients with the APP692 mutation. The neuropathological findings support the diagnosis of AD. Leptomeningial and parenchymal vessels showed extensive deposition of Aβ amyloid protein. Numerous senile plaques consisted of large Aβ amyloid cores, often measuring more than 30 μm in diameter and were surrounded by a fine meshwork of dystrophic neurites. In addition, there were a large number of paired helical filaments in pyramidal neurons and dystrophic neurites. Our findings show that the APP692 mutation leads to morphological abnormalities that are similar to AD, but the morphology of senile plaques is clearly distinct from that described in sporadic and chromosome 14-linked AD patients, in patients with APP717 mutations causing familial, presenile AD and in patients with the APP693 mutation causing HCHWA-D.

Phylogenetic-relationships Among Ascomycetes and Ascomycete-like Yeasts As Deduced From Small Ribosomal-subunit Rna Sequences

The primary structure of the small ribosomal subunit RNA (srRNA) molecule of the type strains of the ascosporogenous yeasts Debaryomcyes hansenii, Pichia anomala (synonym: Hansenula anomala), Pichia membranaefaciens, Schizosaccharomyces pombe, Zygosaccharomyces rouxii and Dekkera bruxellensis was determined. The srRNA sequences were aligned with previously published sequences from fungi, including those of 5 candida species, and an evolutionary tree was inferred The srRNA results were compared with chemotaxonomic criteria, e.g. the coenzyme Q system. The heterogeneity of the genera Candida and Pichia is clearly reflected by the srRNA analysis.

Identification of caspases that cleave presenilin-1 and presenilin-2 - Five presenilin-1 (PS1) mutations do not alter the sensitivity of PS1 to caspases.

Mutations in the presenilin (PS) genes PS1 and PS2 are involved in Alzheimers disease (AD). Recently, apoptosis‐associated cleavage of PS proteins was identified. Here we demonstrate that PS1 as well as PS2 are substrates for different members of the caspase protein family. Remarkably, the caspases acting on PS1 could be subdivided in two groups. One group, containing caspase‐8, ‐6 and ‐11, cleaved PS1 after residues ENDD329 and to a lesser extent after residues AQRD341. A second group consisting of caspase‐3, ‐7 and ‐1 acted uniquely on AQRD341. Importantly, these two cleavage sites were also recognized by caspases in the C‐terminal PS1 fragment produced by constitutive proteolysis. In decreasing order of activity, caspase‐8, ‐3, ‐1, ‐6 and ‐7 proteolysed PS2 at the recognition site D326SYD329. Caspase‐8 and ‐3 exhibited the highest proteolytic activity on both PS1 and PS2. PS1 and PS2 were not hydrolyzed by caspase‐2 and PS2 also not by caspase‐11. None of five missense mutations affected the sensitivity of PS1 to caspase‐mediated cleavage. This suggests that AD pathogenesis associated with PS1 missense mutations cannot be explained by a change in caspase‐dependent processing.

Evolution of basidiomycetous yeasts as deduced from small ribosomal subunit RNA sequences

Complete small ribosomal subunit RNA sequences were used to infer the relationship between several basidiomycetous yeasts, and to resolve the evolutionary position of the basidiomycetes among the fungi. The sequences were determined for Rhodosporidium toruloides (anamorph Rhodotorula glutinis), Filobasidiella neoformans (anamorph Cryptococcus neoformans), Trichosporon cutaneum, Bullera alba and Sporobolomyces roseus. The sequence of Leucosporidium scottii (anamorph formerly named Candida scottii) srRNA has already been published previously (Hendriks et al., J. Mol. Evol. 32, 167-177 (1991)). Using a tree construction program based on a distance matrix, a phylogenetic tree was constructed for all hitherto known fungal srRNA sequences, oomycetes and slime moulds not included. It showed the ascomycetes and the basidiomycetes to be sister groups, probably evolved from a zygomycete-like ancestor and diverged from each other about 840 Myr ago. Among the basidiomycetes, two clearly distinct groups can be recognized, one formed by the teliospore forming species (Rhodosporidium toruloides and Leucosporidium scottii), and the asexual yeast Sporobolomyces roseus, and the other formed by the non-teliospore forming species Filobasidiella neoformans and the asexual yeasts Bullera alba and Trichosporon cutaneum.

Phylogenetic analysis of five medically important Candida species as deduced on the basis of small ribosomal subunit RNA sequences

The classification of species belonging to the genus Candida Berkhout is problematic. Therefore, we have determined the small ribosomal subunit RNA (srRNA) sequences of the type strains of three human pathogenic Candida species; Candida krusei, C. lusitaniae and C. tropicalis. The srRNA sequences were aligned with published eukaryotic srRNA sequences and evolutionary trees were inferred using a matrix optimization method. An evolutionary tree comprising all available eukaryotic srRNA sequences, including two other pathogenic Candida species, C. albicans and C. glabrata, showed that the yeasts diverge rather late in the course of eukaryote evolution, namely at the same depth as green plants, ciliates and some smaller taxa. The cluster of the higher fungi consists of 10 ascomycetes and ascomycete-like species with the first branches leading to Neurospora crassa, Pneumocystis carinii, Candida lusitaniae and C. krusei, in that order. Next there is a dichotomous divergence leading to a group consisting of Torulaspora delbrueckii, Saccharomyces cerevisiae, C. glabrata and Kluyveromyces lactis and a smaller group comprising C. tropicalis and C. albicans. The divergence pattern obtained on the basis of srRNA sequence data is also compared to various other chemotaxonomic data.

Proteolytic processing of presenilin-1 (PS-1) is not associated with Alzheimer's disease with or without PS-1 mutations.

Cerebral presenilin‐1 protein (PS‐1) is normally composed of the amino‐terminal fragment (NTF) with M r 28 kDa and the carboxy‐terminal fragment (CTF) with 18 kDa. We analyzed human PS‐1 in brains with early‐onset familial Alzheimers disease (FAD) with and without PS‐1 mutations to study whether mutated PS‐1 was abnormally metabolized. Cerebral PS‐1 were found to be cleaved into two fragments of NTF and CTF independently of the occurrence of PS‐1 mutation in human brains. A small portion of PS‐1 was recently found to suffer another processing by caspase‐3, an apoptosis‐related cysteine protease. In contrast to the recent finding that the Volga‐German mutation on presenilin‐2 (PS‐2) affects the increasing caspase‐3 PS‐2 fragment, the PS‐1 mutation did not cause a significant change in PS‐1 fragmentation. We conclude that PS‐1 fragmentation and other (probably caspase‐3‐mediated) digestion following apoptosis occur independently of PS‐1 mutations.

The 18S ribosomal RNA sequence of the sea anemone Ammonia sulcata and its evolutionary position among other eukaryotes

Evolutionary trees based on partial small ribosomal subunit RNA sequences of 22 metazoa species have been published [(1988) Science 239, 748‐753]. In these trees, cnidarians (Radiata) seemed to have evolved independently from the Bilateria, which is in contradiction with the general evolutionary view. In order to further investigate this problem, the complete srRNA sequence of the sea anemone Ammonia sulcata was determined and evolutionary trees were constructed using a matrix optimization method. In the tree thus obtained the sea anemone and Bilateria together form a monophyletic cluster, with the sea anemone forming the first line of descent of the metazoan group.