A novel binding assay for detection of 14-3-3 protein in cerebrospinal fluid from Creutzfeldt-Jakob patients.

Here we describe a simple and fast microplate assay for 14-3-3 protein, which is based on the binding of 14-3-3 protein to a chemically synthesized peptide containing the phosphorylated recognition sequence of this protein. The peptide is covalently bound to maleimide-activated microplates through reaction of the sulfhydryl group of a N-terminal cysteine residue linked to the peptide. The peptide- 14-3-.3 complexes formed are detected using--as a second recognition principle--an anti-14-3-3 protein antibody. This assay was used for measurement of 14-3-3 protein in brain (bovine) and in cerebrospinal fluid from patients with Creutzfeldt-Jakob disease (CJD) and control cases with diseases other than CJD. This novel assay allowed also the detection of low 14-3-3 protein levels present in cerebrospinal fluid, without further concentration, in contrast to conventional immunoblotting or immunoassay procedures.

Suppression of PrP(Sc)- and HIV-1 gp120 induced neuronal cell death by sulfated colominic acid.

The scrapie prion protein (PrP(Sc)) has been shown to induce apoptosis of rat cortical neurons in vitro. Here we demonstrate that the toxic effect displayed by PrP(Sc) can be blocked by sulfated colominic acid (polymer of N-acetylneuraminic acid). This compound acts neuroprotectively at a concentration of > or = 0.3 microg/ml when preincubated with the neurons or PrP(Sc). Rat cortical cells also undergo apoptosis after incubation with the HIV-1 coat protein gpl20 in vitro. This effect was abolished also by sulfated colominic acid when preincubated with the cells or gpl20. Addition of 0.3 microg/ml of compound resulted in an increase in cell viability by about 1.6-1.9-fold compared to cultures incubated for 18 h with 30 ng/ml of PrP(Sc) or 20 ng/ml of gpl20 alone (containing about 40% viable cells). Sulfated colominic acid does not act as antagonist of NMDA receptor channels at concentrations of up to 3 microg/ml when co-administered with 100 microg/ml of NMDA. It displayed a strong cytoprotective effect on human T lymphoblastoid CEM cells exposed to HIV-1; a 50% protection occurred after preincubation of the cells with 0.43 microg/ml of compound. At the same concentration, the compound caused an inhibition of HIV-1-induced syncytium formation. Sulfated colominic acid may be a promising compound for treatment of dementia caused by PrP(Sc) and HIV-1 infections.

Anti-HIV-1 activity of inorganic polyphosphates.

Human blood plasma, serum, peripheral blood mononuclear cells, and erythrocytes contain significant amounts of inorganic polyphosphates (ranging from 53 to 116 microM, in terms of phosphate residues). Here we demonstrate that at higher concentrations linear polyphosphates display cytoprotective and antiviral activity. Sodium tetrapolyphosphate and the longer polymers, with average chain lengths of 15, 34, and 91 phosphate residues, significantly inhibited human immunodeficiency virus type 1 (HIV-1) infection of cells in vitro at concentrations > or = 33.3 microg/ml (> or = 283-324 microM phosphate residues), whereas sodium tripolyphosphate was ineffective. In the tested concentration range, these compounds had no effect on cell growth. The longer-chain polyphosphates (polyphosphates with mean chain lengths of 15 and 34) but not sodium tripolyphosphate and sodium tetrapolyphosphate also inhibited HIV-1-induced syncytium formation at a concentration of 160 microg/ml (1.51-1.54 mM phosphate residues). The results obtained with the syncytium assay and by cell-virus binding experiments indicate that the anti-HIV effect of these nontoxic polyanions may be caused by binding of the compounds to both the host cell surface and the virus, thereby inhibiting adsorption of the virus. Competition experiments revealed that binding of [32P]polyphosphate to Molt-3 cells was only partially inhibited by the antibody OKT4A.

Flupirtine protects both neuronal cells and lymphocytes against induced apoptosis in vitro: implications for treatment of AIDS patients.

In the present study we demonstrate that flupirtine, an already clinically used, centrally acting, non-opiate analgesic agent, protects rat cortical neurons against HIV-gp120 induced apoptotic cell death. The drug was active at concentrations between 1 and 10 microg/ml. Furthermore we show inhibition of in vitro induced apoptosis in human blood mononuclear cells, using flupirtine. Induced apoptosis in peripheral blood mononuclear cells from healthy individuals and HIV-1 infected patients was reduced to approximately 50% after in vitro preincubation with flupirtine at concentrations between 0.1 and 10 microg/ml. The anti-apoptotic effect of flupirtine was restricted to CD3+ lymphocytes and in particular to CD4+ cells. Flupirtine does not affect uninduced apoptosis in human lymphocytes in vitro. The selective potential of flupirtine to reduce apoptosis without influencing uninduced apoptosis may qualify this compound as a potential drug in the therapy of HIV-1 infected patients.

Molecular cloning and sequence analysis of two cDNAs coding for putative anionic trypsinogens from the colonial Urochordate Botryllus schlosseri (Ascidiacea).

Botryllus schlosseri is a colonial marine invertebrate that belongs to the subphylum Urochordata. Previously we analyzed the activity of a serine protease in this species, and cloned a tunicate chymotrypsin-like molecule. In the present study we further analyzed the protease activity of this animal, and found biochemical evidence also for specific trypsin-like activity. Subsequently we utilized a degenerate polymerase chain reaction (PCR) primer to clone two B. schlosseri cDNAs coding for two different putative trypsinogens, each 243 amino acids long, that differ within the coding region in 42 amino acids and 99 nucleotides. Both clones feature the characteristics of animal anionic trypsinogens. Sequence analysis of the tunicate putative trypsinogens revealed the invertebrate characteristics of three disulfide bridges, and higher similarity to invertebrate than to vertebrate trypsinogens. We therefore propose that the typical characteristics of vertebrate trypsinogens evolved after the divergence of Urochordates and Cephalochordates.