Roberto Comolli

Increased plasma levels of soluble CD40, together with the decrease of TGFβ1, as possible differential markers of Alzheimer disease

Alzheimers disease (AD) is a progressive neurodegenerative illness and the most frequent cause of dementia in the elderly. The identification of activated microglia within neuritic plaques, coupled with the presence of numerous inflammatory proteins, suggests that inflammation is an integral part of the pathogenetic process in AD. In the present paper we have investigated the levels of circulating inflammatory mediators as potential AD biomarkers concentrating essentially on (a) soluble CD40 (sCD40), a member of the tumor necrosis factor receptor superfamily lacking the membrane-associated endodomain by alternative splicing, and (b) transforming growth factor (TGF)-beta 1, a cytokine deeply involved in AD and playing a protective role on CNS. Decrease of TGF-beta1 in AD patients could enhance the effects of pro-inflammatory cytokines produced by activated microglia as well as the expression of factors, such as the CD40/CD40 ligand complex, by microglia and astrocytes. Total venous blood samples were obtained from 33 patients with clinical diagnosis of possible late-onset AD, 40 healthy age-matched and 11 healthy young individuals. A significant increase of sCD40 levels plasma of AD patients versus healthy controls was measured, concomitantly with a decrease in TGF-beta1 concentration. These variations, however, showed no correlation with the expression of ApoE epsilon 4 allele, which was determined in order to assess the different frequency of this risk factor between AD and control groups. Since no comparable modifications were detected in patients affected by Parkinsons disease or non-AD-based dementia, we propose that sCD40 and TGF-beta1 plasma levels might represent possible differential biomarkers of AD, and be useful pre-mortem to support the clinical diagnosis of late-onset AD.

Decrease of TGF-β1 plasma levels and increase of nitric oxide synthase activity in leukocytes as potential biomarkers of Alzheimer's disease

Abstract A variety of inflammatory proteins have been identified in brains of Alzheimers disease (AD) patients, including inflammatory cytokines, acute phase proteins and complement components. In the present paper we have investigated the levels of circulating inflammatory mediators as potential biomarkers of this disease, concentrating mostly on transforming growth factor beta (TGF-β1) in plasma and on nitric oxide synthase (NOS) activity in leukocytes. Plasma and leukocytes were isolated from 48 sporadic AD and 23 healthy control subjects of same age and sex. Since α2-Macroglobulin (α2M), an acute phase protein possibly involved in AD, is an important modulator of TGF-β1 activity, binding and targeting this cytokine to its appropriate site of action, we have investigated the possible complex between TGF-β1 and α2M in plasma of the same subjects. The results demonstrate a significant reduction of TGF-β1 levels in plasma of AD patients. A complex between α2M and TGF-β1 occurred in AD as well as healthy elderly control subjects, however, with no significant differences. Moreover, α2M appeared to bind only the inactive form of this cytokine. In contrast, NOS activity increased significantly in leukocytes of AD patients. Therefore, we suggest the combined determination of TGF-β1 in the plasma and of NOS activity in the leukocytes as biomarkers of AD.

Protein turnover of the lysosomal and mitochondrial fractions of rat liver during aging

In vivo studies on protein turnover and amino acid incorporation in the mitochondrial, lysosomal and crude microsomal fractions of rat liver have been conducted by a double isotope procedure, using 14C-lysine and 3H-lysine, in 1, 4–6, 12 and 20–26 month-old male animals. The apparent turnover rate of mitochondrial proteins, purified from lysosomal contamination by gentle digitonin treatment, and estimated by their 3H/14C ratios, is slower than that of the other protein fractions examined and does not vary appreciably with age. The apparent turnover of lysosomal proteins, purified from mitochondrial and microsomal contamination by centrifugation in sucrose density gradients, appears higher than that of the mitochondrial fraction and similar to that of the crude microsomal proteins. Both these fractions show apparent diminutions of protein turnover with age that are statistically significant when differences are compared between young and senescent animals. The degree of amino acid incorporation into protein is also affected by age. It is significantly lower in young rats, reaches the maximum in adult 12 month-old animals and decreases slightly in the older group.

Age-dependent modulation of PKC isoforms and NOS activity and expression in rat cortex, striatum, and hippocampus

Recently, PKC has been shown to play a pivotal role in physiological brain functions, connected with the memorizing processes and their correspondent progressive decline with brain aging. We have studied the age-dependent changes of PKC isoforms activity in connection with NOS expression and activity in specific brain areas such as hippocampus, cortex and striatum. Starting from middle aged rats, a significant inactivation of c-PKC isoforms occurred, with respect to young animals, in all the brain areas analysed. However, in middle aged animals, no significant changes in the protein level of the main PKC isoforms expressed in brain were demonstrated. Moreover, in the hippocampus and in the cortex of middle aged rats, an increased level of NOS activity--a substrate of PKC whose phosphorylation by the kinase inhibits NOS activity--has been demonstrated, reaching the same level that occurs in striatum. However, only in the hippocampus, deeply implicated in learning and memory functions, an increase of nuclear c-PKC activity and of i- and n-NOS mRNA levels was shown. Taken together, these results indicate that down-regulation of c-PKC activity occurring in middle aged rats, leads to higher levels of NO that may contribute to cell damage and to alter the neuronal physiological functions as described in older animals. Moreover, in the hippocampus, our results suggest a relationship between the translocation of c-PKC to the nucleus and the enhancement of the expression of i- and n-NOS.

ACTIVATION OF PROTEIN KINASE C-ALPHA ISOFORM IN MURINE MELANOMA CELLS WITH HIGH METASTATIC POTENTIAL

AbstractMetastasis is a multistep process in which protein kinase C (PKC) appears to be significantly involved. We analysed the activity and expression of classical (α, β, γ) and novel PKC

Overexpression of novel protein kinase C delta in BL6 murine melanoma cells inhibits the proliferative capacity in vitro but enhances the metastatic potential in vivo.

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Seasonal changes and temperature-dependent accumulation of polycyclic aromatic hydrocarbons in high-altitude soils.

isoforms in B16-F1 and B16-BL6 melanoma cells maintained under different culture conditions in vitro. We used high and low concentrations of tyrosine and phenylalanine in different media (DMEM or RPMI 1640 respectively) that affect the metastatic potential and also the proliferative capacity of the cells. We also tested a weakly metastatic amelanotic B78-H1 melanoma cell line which is unaffected by the different culture conditions. In both B16 melanoma cell lines activation of PKC α (without increased expression) occurred under growth conditions permissive of metastasis (DMEM). In contrast, the weakly metastatic amelanotic B78-H1 cell line showed a substantial inactivation of this isoform in the two different culture media, suggesting a specific involvement of PKC α in the metastatic process. Moreover, in B16 melanoma cells, novel PKC