An amyloid protein that precipitates in the cerebral vessel walls of Dutch patients with hereditary cerebral hemorrhage with amyloidosis is similar to the amyloid protein in vessel walls and senile plaques in brains of patients with Alzheimer's disease, Down syndrome, and sporadic cerebral amyloid angiopathy. Cloning and sequencing of the two exons that encode the amyloid protein from two patients with this amyloidosis revealed a cytosine-to-guanine transversion, a mutation that caused a single amino acid substitution (glutamine instead of glutamic acid) at position 22 of the amyloid protein. The mutation may account for the deposition of this amyloid protein in the cerebral vessel walls of these patients, leading to cerebral hemorrhages and premature death.

No abstract

Alzheimer's disease is characterized by the extracellular deposition of ␤-amyloid peptide (A␤) in cerebral plaques and evidence is accumulating that amyloid is neurotoxic. A␤ is derived from the ␤-amyloid precursor protein (APP). Proteolytic processing of APP by the enzyme, ␤-secretase, produces the N terminus of A␤, and releases a secreted ectodomain of APP (␤-s-APP). To develop animal models for measuring ␤-secretase activity in specific brain cells in vivo, we have targeted the expression of the full-length human APP to either neurons or astrocytes in transgenic mice using the neuronspecific enolase (NSE) promoter or a modified glial fibrillary acidic protein (GFAP) gene, respectively. The APP cDNAs expressed were mutated (KM to NL at 670/ 671) to encode amino acid substitutions that enhance amyloidogenic processing in vitro. Western analyses revealed abundant production of ␤-s-APP in the brains of NSE-APP mice and enzyme-linked immunosorbent assay analyses showed production of A␤ in fetal primary mixed brain cultures and brain homogenates from these transgenic animals. Because the NSE promoter drives expression primarily in neurons, this provides in vivo evidence that the ␤-secretase cleavage necessary for generation of ␤-s-APP and A␤ is efficiently performed in neurons. In contrast, only little ␤-s-APP was detected in brain homogenates of GFAP-APP mice, indicating that astrocytes show very little ␤-secretase activity in vivo. This provides strong in vivo evidence that the major source of A␤ in brain is from neurons and not from astrocytes.The extracellular deposition of ␤-amyloid peptide (A␤) 1 in senile plaques is an early and invariant feature of Alzheimer's disease (AD). This 39 -43-amino acid peptide is the major component of plaques and is proteolytically processed from the ␤-amyloid precursor protein (APP) (1, 2). APP is expressed in all tissues, and the relative amount of A␤ processed from APP varies in different cell types in culture (3-5). The cellular source of A␤ deposited into plaques in the brain is unknown. Mutations in APP are responsible for some forms of familial AD, supporting the hypothesis that APP and A␤ are central to the disease process (6). Missense mutations immediately Nterminal to the A␤ region of APP 2 lead to a 5-10-fold enhancement of A␤ produced from APP in vitro, strongly supporting the role of A␤ in the development of AD in this family (7,8). Other families, with mutations at the 717 position of APP, have been shown to produce increased amounts of the more amyloidogenic 42-amino acid form of A␤ from APP (9). These findings suggest that factors governing the metabolic processing of APP play a direct pathogenic role in Alzheimer's disease.The majority of APP is cleaved in the middle of the A␤ region, releasing a secreted ectodomain containing the first 16 amino acids of A␤ (␣-s-APP). This processing, mediated by an unidentified enzymatic activity termed "␣-secretase" precludes A␤ formation (10). In an alternative pathway, cleavage between Met 671 and Asp 672 by a likewise unid...

A patient is described with acute myelocytic leukemia refractory to conventional therapy, who also became highly resistant to methotrexate (MTX) after repeated courses of this drug. Leukemia cells from this patient were found to contain an elevated level of dihydrofolate reductase (DHFR) activity, with no change in the affinity of the enzyme for MTX. A sensitive "dot blot" assay revealed a fourfold increase in the gene copy number of DHFR. Southern blot analysis with a human DHFR cDNA probe confirmed this increase in the gene copy number, and demonstrated a similar restriction pattern with Eco R1, Hind III, and Pst 1 as seen with a highly amplified human leukemia cell line, K562. Additional DHFR fragments were detected, not seen in the K562 blot, suggesting the presence of pseudogenes, or a result of gene rearrangements occurring as part of the amplification process. Resistance to MTX in this patient was therefore ascribed to gene amplification and overproduction of DHFR.

We have shown previously that cDNAs for the Ml and M2 subunits of ribonucleotide reductase, ornithine decarboxylase (ODC), and p5–8, a 55,000-Dalton protein, hybridize to amplified genomic sequences in a highly hydroxyurea-resistant hamster cell line. We have extended these observations to include two additional, independently isolated, hydroxyurea-resistant cell lines: SC8, a single-step hamster ovary cell line, and KH450, a multistep human myeloid leukemic cell line, have also undergone genomic amplification for sequences homologous to ODC and p5–8 cDNAs. However, neither SC8 nor KH450 contains amplified genomic sequences homologous to an Ml cDNA probe. A panel of mouse-hamster somatic cell hybrids was used to map sequences homologous to Ml, M2, ODC, and 5–8 cDNAs in the hamster genome. The M2, ODC, and p5–8 cDNAs hybridized to DNA fragments that segregated with hamster chromosome 7. In contrast, Ml cDNA hybridized to DNA fragments that segregated with hamster chromosome 3. These data suggest that the genes RRM2, (M2), ODC, and p5–8, but not RRM1 (Ml), are linked and may have been co-amplified in the selection of the hydroxyurea-resistant hamster and human cell lines.