Spontaneous Aggregation and Cytotoxicity of the β-Amyloid Aβ1–40: A Kinetic Model

The time dependency of the spontaneous aggregation of the fibrillogenic β-Amyloid peptide, Aβ^1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Aβ antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 μM amyloid peptide. The anti–Aβ antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length Aβ was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the Aβ^1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of Aβ^1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of Aβ^1–40 in vitro and possibly in vivo.     

D8S7 is consistently deleted in inverted duplications of the short arm of chromosome 8 (inv dup 8p)

Ten patients with inverted duplication of 8p (inv dup 8p) were studied with cytogenetic, biochemical and molecular techniques. The duplication for the region 8p12-p22 was always associated with a deletion of the locus D8S7 (mapped in 8p23.1) as demonstrated with the probe pSW50 by both in situ hybridization and Southern blot. Restriction fragment length polymorphisms detected by probes pSW50 (1 case) and by pG2LPL35 (locus LPL) (two cases) were informative as to a maternal origin of the anomaly. The activity of glutathione reductase, whose gene maps in the duplicated region at 8p21.1, was increased in all patients. The recognizable phenotype of inv dup 8p includes neonatal hypotonia, prominent forehead, large mouth with everted lower lip, abnormally shaped large ears, brain malformations and severe mental retardation. Our findings indicate that the chromosome rearrangement is homogeneous at least for the presence of the deletion and support the hypothesis of a common mechanism of origin.     

Synergistic Stimulation of Interleukin 6 Release and Gene Expression by Phorbol Esters and Interleukin 1β in Rat Cortical Astrocytes: Role of Protein Kinase C Activation and Blockade

Abstract: The involvement of protein kinase C and its interaction with interleukin 1β in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short-term phorbol 12-myristate 13-acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12-myristate 13-acetate enhanced concentration- and time-dependently interleukin 6 release. This stimulatory action of phorbol 12-myristate 13-acetate was significantly reduced by staurosporine, by calphostin C, and by the desensitization of protein kinase C. Interleukin 1β increased interleukin 6 release in a concentration-related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1β stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1β (3 ng/ml) and phorbol 12-myristate 13-acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurosporine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C. Interleukin 1β stimulated interleukin 6 secretion via a mechanism that is also negatively modulated by a protein kinase C isoform or isoforms sensitive to staurosporine and desensitization. Finally, we showed that interleukin 1β and phorbol 12-myristate 13-acetate synergistically stimulated interleukin 6 release and its gene expression, operating in a manner insensitive to protein kinase C blockers and slightly reduced by protein kinase C desensitization.     

Liver fatty acid-binding protein in two cases of human lipid storage

Summary FABPs in the various tissues play an important role in the intracellular fatty acid transport and metabolism. Reye's syndrome (RS) and multisystemic lipid storage (MLS) are human disorders characterized by a disturbance of lipid metabolism of unknown etiology. We investigated for the first time L-FABP in these two conditions. Affinity purified antibodies against chicken L-FABP were raised in rabbits, and found to cross-react specifically with partially purified human L-FABP. L-FABP content in liver samples of two patients with RS and MLS was investigated by immuno-histochemistry, SDS-PAGE and ELISA. L-FABP immuno-histochemistry showed increased reactivity in the liver of RS patient and normal reactivity in MLS liver. L-FABP increase in RS liver was confirmed by densitometry of SDS-PAGE and ELISA method. By these two methods the increase amounted to 180% and 199% (p < 0.02), respectively, as compared to controls. A possible role of L-FABP in the pathogenesis of RS is discussed.     

Singular value decomposition analysis of protein sequence alignment score data.

One of the standard tools for the analysis of data arranged in matrix form is singular value decomposition (SVD). Few applications to genomic data have been reported to date mainly for the analysis of gene expression microarray data. We review SVD properties, examine mathematical terms and assumptions implicit in the SVD formalism, and show that SVD can be applied to the analysis of matrices representing pairwise alignment scores between large sets of protein sequences. In particular, we illustrate SVD capabilities for data dimension reduction and for clustering protein sequences. A comparison is performed between SVD-generated clusters of proteins and annotation reported in the SWISS-PROT Database for a set of protein sequences forming the calycin superfamily, entailing all entries corresponding to the lipocalin, cytosolic fatty acid-binding protein, and avidin-streptavidin Prosite patterns.