Involvement of rat hippocampal astrocytes in ß-amyloid-induced angiogenesis and neuroinflammation.

Although Alzheimer's disease (AD) is considered a neurodengenerative disorders, in the last few years a large amount of evidence has suggested that it is also a vascular pathology characterized by increased capillary density and expression of angiogenic factors. In AD the endothelium degenerates, promoting local neuroinflammation and activation of brain endothelium, perivascular microglia, pericytes, astrocytes. Excess tumor necrosis factor (TNF) in the cerebrospinal fluid (CSF), at a concentration of 25 times higher than in the control group, has been demonstrated in AD. Recent studies provide evidence that treatment with TNF-α antagonists may result in a rapid cognitive improvement in AD patients. In the present work we investigated the role of astrocytes in AD angiogenesis and neuroinflammation by means of conditioned media of untreated and Aß-treated rat hippocampal astrocytes (RHAs) on rat microvascular endothelial cells (RCECs). The results demonstrated that RHA media increase RCEC proliferation and capillary-like structure formation. Moreover RHAs secrete IL-1ß and, only after the Aß1-42 treatment, TNF-α promotes RCEC release of IL-1ß, IL-6 and TNF-α. The removal of IL-1ß, TNF-α and/or VEGF, a strong angiogenic inducer highly over-expressed in AD brains, by means of specific antibody-coated beads in RHA media affects RCEC release of IL-1ß, IL-6 and TNF-α. We hypothesised that astrocytes contribute to AD angiogenesis and neuroinflammation by the direct release of pro-inflammatory cytokines. The effect of an anti-inflammatory agent, such as etanercept, decreased RCEC in vitro cytokine release. This could be compared to the effect found in our experiments with antibody anti TNF-α-coated beads.

Electrophoretic analysis of mammalian hair keratins.

Keratins extracted from the hair of several mammals were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). By SDS-PAGE, most differences between species were detected in the position and intensity of polypeptide bands with MW in the range 30-10 KDa (high sulphur keratins, hair matrix proteins, HMP). By IEF, differences were found in the intensity of polypeptide bands in the acidic pH range (< 5.1). Electrophoretic techniques could therefore have a taxonomic usefulness.

Electrophoretic analysis of non-human primates hair keratin.

Keratin characterization through electrophoretic techniques has been used for species identification in forensic science and in taxonomic studies. In the present work, protein components solubilized from hair of non-human primates were evaluated to investigate whether there is any species-specific pattern in an evolutionary perspective, by grossly comparing hair native keratins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Extracted hair keratins for all specimens were separated by SDS-PAGE into 2-3 polypeptide bands with apparent MW in the range 39-54 kDa and into 3-7 polypeptide bands with apparent MW in the range 10-35 kDa. With this technique it was possible to distinguish different suborders, different families of the same suborder, and, sometimes, different genera from the same family. On the contrary, it was not possible to distinguish different species of the same genus and different specimens of the same species. With IEF, extracted hair keratins were separated into about 30 polypeptide bands with pI values in the range of pH 3.9-7.7. IEF discriminates poorly between different samples. Only in specimens from Papio genus did we find an additional polypeptide band. In conclusion, we found that the differences between electrophoretic patterns are largest for animals that are not closely related while specimens of the same species have the same patterns.

Electrophoretic analysis of human hair keratins.

Non S-carboxymethylated human keratins, extracted from the hair of individuals from 3 Italian families, were characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF), after the separation of hair fibrous proteins (HFP) and hair matrix proteins (HMP) by gel filtration chromatography. By SDS-PAGE, HFP were separated into 3 polypeptide bands with apparent molecular weight (MW) 52 KDa, 44KDa and 40 KDa; by IEF they were separated into several polypeptide bands with apparent isoelectric point (pI) in the pH range 7.4-4.0. By IEF, HMP were separated into several polypeptide bands with apparent pI in the pH range 7.4-5.4. It was not possible to characterize HMP by SDS-PAGE since their concentration, obtained after gel filtration chromatography, was very low. However, total extracted hair keratins were separated by SDS-PAGE into the polypeptides of HFP and into two bands with apparent MW 16 KDa, and 12 KDa. It was not possible to point out any interindividual variability in the electrophoretic patterns, except for one woman, for whom cosmetic treatment of hair could not be excluded. Finally, electrophoretic patterns for subjects of the same family are more similar than for unrelated subjects. Electrophoretic analysis of fractionated hair keratins could be useful for a better characterization of these polypeptides in genetic studies.

Analysis of serum apolipoprotein A-I in elderly non-insulin-dependent diabetic patients.

Quantitative and qualitative modifications of plasma apolipoproteins could provide additional information in predicting coronary heart disease (CHD) in non-insulin-dependent diabetes mellitus (NIDDM). We examined 17 men and 15 women aged over 65 years affected by NIDDM. Alcohol intake, smoking habits, antidiabetic therapy, body weight, stature, and body mass index were considered. Furthermore, serum lipids and apolipoprotein A-I and B levels were determined. Apo A-I was characterized by isoelectric focusing (IEF) and immunoblotting. The men were heavier and taller than the women. The women had significantly higher serum levels of total cholesterol, HDL-cholesterol, and apo A-I than the men. The levels of HDL-cholesterol, apo A-I and B were significantly influenced by an interaction between sex and the duration of diabetes. An invariant pattern was observed for apo A-I isoforms in samples from diabetic subjects of both sexes. The percentage distribution of serum apo A-I isoforms determined by densitometry is quite different from that previously reported in the literature for the general population. Quantitative modification in apo A-I isoforms could be due to the specific pathology and these variations could partly explain reduced levels of HDL-cholesterol in diabetic patients.