Amyloid b-peptide (Ab) 1)42 oligomers have recently been discussed as intermediate toxic species in Alzheimer's disease (AD) pathology. Here we describe a new and highly stable Ab 1)42 oligomer species which can easily be prepared in vitro and is present in the brains of patients with AD and Ab 1)42 -overproducing transgenic mice. Physicochemical characterization reveals a pure, highly water-soluble globular 60-kDa oligomer which we named 'Ab 1)42 globulomer'. Our data indicate that Ab 1)42 globulomer is a persistent structural entity formed independently of the fibrillar aggregation pathway. It is a potent antigen in mice and rabbits eliciting generation of Ab 1)42 globulomer-specific antibodies that do not cross-react with amyloid precursor protein, Ab 1)40 and Ab 1)42 monomers and Ab fibrils. Ab 1)42 globulomer binds specifically to dendritic processes of neurons but not glia in hippocampal cell cultures and completely blocks long-term potentiation in rat hippocampal slices. Our data suggest that Ab 1)42 globulomer represents a basic pathogenic structural principle also present to a minor extent in previously described oligomer preparations and that its formation is an early pathological event in AD. Selective neutralization of the Ab globulomer structure epitope is expected to have a high potential for treatment of AD. Keywords: Alzheimer's disease, amyloid b-peptide, hippocampal neurons, long-term potentiation, oligomers, polymerization. Abbreviations used: Ab, amyloid b-peptide; AD, Alzheimer's disease; ADDL, amyloid b-peptide-derived diffusible ligand; AFM, atomic force microscopy; APP, amyloid precursor protein; CHO, chinese hamster ovary; CL, cross-linked; CSF, cerebrospinal fluid; DAPI, 4¢,6-Diamidino-2-phenylindole; DIV, days in vitro; EPSPs, excitatory postsynaptic potentials; fEPSP, field excitatory postsynaptic potential; GFAP, glial fibrillary acidic protein; HFIP, 1,1,1,3,3,3 hexafluoro-2-propanol; HFS, high-frequency stimulation; LTP, long-term potentiation; MAP2, microtubule associated protein-2; NBT/BCIP, Nitro blue tetrazolium chloride/5-Bromo-4-chloro-3-indolyl phosphate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PSD-95, postsynaptic density protein 95; RT, room temperature; sAPPa, soluble amyloid precursor protein alpha; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; TBST, 0.05% Tween 20 in Tris-buffered saline.

Alzheimer's disease (AD) is a neurodegenerative disorder that is linked to the presence of amyloid beta-peptides that can form insoluble fibrils or soluble oligomeric assemblies. Soluble forms are present in the brains and tissues of Alzheimer's patients, and their presence correlates with disease progression. Long-lived soluble forms can be generated in vitro by using small amounts of aliphatic hydrocarbon chains of detergents or fatty acids in preparations of amyloid beta-peptides. Using NMR, we have characterized soluble oligomers of Abeta preglobulomer and globulomer that are stable and alter synaptic activity. The NMR data indicate that these soluble forms have a mixed parallel and antiparallel beta-sheet structure that is different from fibrils which contain only parallel beta-sheets. Using the structural data, we engineered a disulfide bond into the soluble Abeta globulomer to give a "new" soluble antigen that is stable, homogeneous, and binds with the same affinity to selective antibodies as the parent wt globulomer.

Tumour necrosis factor (TNF) was first described as a factor in the serum of mice injected with tubercle bacilli (BCG) and several days later with lipopolysaccharide (LPS). The gene encoding TNF has recently been cloned and pure recombinant human TNF is now available. TNF is known for its in vivo antitumour effect and in vitro cytotoxicity on certain transformed cell lines. Similarities in amino acid sequence and biological activity to lymphotoxin and cachectin have been reported, and very recently a growth-factor-like activity on diploid fibroblasts was observed. There is no similarity between these proteins and interferons (IFNs), which are also induced during in vivo induction of TNF. Here we describe the antiviral activity of pure recombinant human TNF in a typical in vitro antiviral assay which we discovered while investigating the possible role of TNF as an inducer of IFN.

Abnormal accumulation of soluble oligomers of amyloid ␤ (A␤) is believed to cause malfunctioning of neurons in Alzheimer's disease. It has been shown that A␤ oligomers impair synaptic plasticity, thereby altering the ability of the neuron to store information. We examined the underlying cellular mechanism of A␤ oligomer-induced synaptic modifications by using a recently described stable oligomeric A␤ preparation called "A␤ 1-42 globulomer." Synthetically prepared A␤ 1-42 globulomer has been shown to localize to neurons and impairs long-term potentiation (Barghorn et al., 2005). Here, we demonstrate that A␤ 1-42 globulomer does not affect intrinsic neuronal properties, as assessed by measuring input resistance and discharge characteristics, excluding an unspecific alteration of membrane properties. We provide evidence that A␤ 1-42 globulomer, at concentrations as low as 8 nM, specifically suppresses spontaneous synaptic activity resulting from a reduction of vesicular release at terminals of both GABAergic and glutamatergic synapses. EPSCs and IPSCs were primarily unaffected. A detailed search for the precise molecular target of A␤ 1-42 globulomer revealed a specific inhibition of presynaptic P/Q calcium currents, whereas other voltage-activated calcium currents remained unaltered. Because intact P/Q calcium currents are needed for synaptic plasticity, the disruption of such currents by A␤ 1-42 globulomer may cause deficits in cellular mechanisms of information storage in brains of Alzheimer's disease patients. The inhibitory effect of A␤ 1-42 globulomer on synaptic vesicle release could be reversed by roscovitine, a specific enhancer of P/Q currents. Selective enhancement of the P/Q calcium current may provide a promising strategy in the treatment of Alzheimer's disease.

A membrane-bound protease activity that specifically converts Big endothelin-1 has been purified from bovine endothelial cells (FBHE). The enzyme was cleaved with trypsin and the peptide sequencing analysis confirmed it to be a zinc chelating metalloprotease containing the typical HEXXH (HELTH) motif. RT-PCR and cDNA screens were employed to isolate the complete cDNAs of the bovine and human enzymes. This human metalloprotease was expressed heterologously in cell culture and oocytes. The catalytic activity of the recombinant enzyme is the same as that determined for the natural enzyme. The data suggest that the characterized enzyme represents the functional human endothelin converting enzyme ECE-1.