Obesity is increasing in an epidemic manner in most countries and constitutes a public health problem by enhancing the risk for cardiovascular disease and metabolic disorders such as type 2 diabetes. Owing to the increase in obesity, life expectancy may start to decrease in developed countries for the first time in recent history. The factors determining fat mass in adult humans are not fully understood, but increased lipid storage in already developed fat cells (adipocytes) is thought to be most important. Here we show that adipocyte number is a major determinant for the fat mass in adults. However, the number of fat cells stays constant in adulthood in lean and obese individuals, even after marked weight loss, indicating that the number of adipocytes is set during childhood and adolescence. To establish the dynamics within the stable population of adipocytes in adults, we have measured adipocyte turnover by analysing the integration of 14C derived from nuclear bomb tests in genomic DNA. Approximately 10% of fat cells are renewed annually at all adult ages and levels of body mass index. Neither adipocyte death nor generation rate is altered in early onset obesity, suggesting a tight regulation of fat cell number in this condition during adulthood. The high turnover of adipocytes establishes a new therapeutic target for pharmacological intervention in obesity.

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Low levels of anti-PC could be of importance in SLE. Anti-BSA and anti-PS and low levels of anti-PC could contribute to development of CVD in SLE.

The heat-shock proteins (HSPs) Hsp27 and alphaB-crystallin are up-regulated in Alzheimer's disease (AD), but the extent of this and the consequences are still largely unknown. The HSPs are involved in protein degradation and protection against protein aggregation, and they interact with several cytoskeletal components such as microtubules (MT) and neurofilaments (NF). AD pathology includes aggregated proteins (tau, NF), decreased protein degradation, and cytoskeletal disruption. It is thus of interest to investigate more closely the possible roles of the HSPs in AD pathology. The expressions of Hsp27 and alphaB-crystallin in AD brain samples were significantly increased (by approximately 20% and approximately 30%, respectively) and correlated significantly with phosphorylated tau and NF proteins. To investigate the consequences of increased HSP levels on tau and NF regulation, N2a cells were transfected with Hsp27 or alphaB-crystallin constructs, and overexpression of the HSPs was confirmed in the cells. Increased tau phosphorylation at the Ser262 site in the N2a cells was regulated by Hsp27 overexpression (possibly through p70S6k), whereas the overexpression of alphaB-crystallin resulted in decreased levels of phosphorylated tau, NF, and GSK-3beta. It was also shown that overexpression of HSPs causes an increase in the percentage of cells present in the G(1) phase. The results presented suggest that a cellular defense against dysregulated proteins, in the form of Hsp27 and alphaB-crystallin, might contribute to the cell cycle reentry seen in AD cells. Furthermore, Hsp27 might also be involved in AD pathology by aggravating MT disruption by tau phosphorylation.

Increasing evidence indicates that mitochondrial alterations contribute to the neuronal death in Alzheimer's disease (AD). Presenilin 1 (PS1) and Presenilin 2 (PS2) mutations have been shown to sensitize cells to apoptosis by mechanisms suggested to involve impaired mitochondrial function. We have previously detected active gamma-secretase complexes in mitochondria. We investigated the impact of PS/gamma-secretase on mitochondrial function using mouse embryonal fibroblasts derived from wild-type, PS1-/-, PS2-/- and PS double knock-out (PSKO) embryos. Measurements of mitochondrial membrane potential (DeltaPsim) showed a higher percentage of fully functional mitochondria in PS1-/- and PSwt as compared to PS2-/- and PSKO cells. This result was evident both in whole cell preparations and in isolated mitochondria. Interestingly, pre-treatment of isolated mitochondria with the gamma-secretase inhibitor L-685,458 resulted in a decreased population of mitochondria with high DeltaPsim in PSwt and PS1-/- cells, indicating that PS2/gamma-secretase activity can modify DeltaPsim. PS2-/- cells showed a significantly lower basal respiratory rate as compared to other cell lines. However, all cell lines demonstrated competent bioenergetic function. These data point toward a specific role of PS2/gamma-secretase activity for proper mitochondrial function and indicate interplay between PS1 and PS2 in mitochondrial functionality.

SummaryTP53 is mutated in 10-20% of cases of chronic lymphocytic leukaemia (CLL) and 3-8% of cases of acute myeloid leukaemia (AML). Recently, two classes of compounds that restore the function of p53 in tumours have been described. PRIMA-1 (p53-dependent reactivation and induction of massive apoptosis) restores the wild-type conformation of mutant TP53, whereas RITA (reactivation of p53 and induction of tumour cell apoptosis) increases intracellular levels of p53. We evaluated the effects of RITA alone and in combination with PRIMA-1 or conventional cytostatics on leukaemic cells isolated from AML and CLL patients. AML samples with )17, which are more resistant to daunorubicin and cytarabine compared with samples without )17, were effectively killed by PRIMA-1. RITA, which stabilizes the function of wild-type p53, induced apoptosis in AML cells. In contrast to that seen with PRIMA-1, AML patient samples without )17 were significantly more sensitive to RITA. Similarly, RITA exerted dosedependent apoptosis and cytotoxicity in CLL cells, which was significantly more pronounced in samples without hemizygous TP53 deletion. Notably, a synergistic effect was observed in all CLL samples with RITA and fludarabine in combination. In both AML and CLL cells exposure to RITA resulted in induction of intracellular p53. We conclude that small molecules targeting p53 might be of clinical importance in the future for treating drug-resistant leukaemia.Keywords: PRIMA, RITA, )17, acute myeloid leukaemia, chronic lymphocytic leukaemia. (Xue et al, 2007). Thus, in vivo models provide firm support to the idea of p53 reactivation for treatment of cancer. Reinstating p53 appears to be feasible, since the p53 protein is usually expressed in tumours, although it is functionally inert.Different strategies of p53 rescue for the selective elimination of tumours can be envisioned, depending on the type of p53 inactivation. In tumours carrying wt TP53 its function can be inhibited by HDM2 (MDM2 in mice) (Momand et al, 1992). HDM2 forms a complex with p53 protein by binding to its amino terminus, leading to proteasomal degradation of p53 (Chene, 2003) (Banin et al, 1998). DNA damage decreases the activity of HDM2 and the interaction between HDM2 and p53, which eventually results in stabilization of p53 (Lakin & Jackson, 1999), (Vogelstein et al, 2000) via phosphorylation of both p53 and HDM2 by the protein kinase ATM (Kojima et al, 2005).Recently, two small molecules that can restore the tumour suppressor function of p53 in cancer cells have been described. They were identified after screening of the NCI diversity set chemical library. RITA (reactivation of p53 and induction of tumour cell apoptosis; 2AE5-bis (5-hydroxymethyl-2-thienyl) furan) (Bates & Vousden, 1999) was selected by using a pair of isogenic cell lines with different p53 status. RITA binds to the p53 N-terminal domain and induces its accumulation in tumour cells lines, increasing its half-life by preventing p53-HDM2 interaction. RITA exerts tumour suppression in tumours with ...

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The mechanisms associated with cell death have been an important focus for neurobiology research. In the present study, the methodology of flow cytometry was used to optimize quantification of the toxic effects of tumor necrosis factor-alpha (TNF-alpha), trans-4-hydroxy-2-nonenal (4-HNE), and aged amyloid-beta (Abeta1-42) on rat primary cortical neurons. The fluorescent dyes annexin V-FITC and propidium iodide (PI) were used to identify populations of viable, early apoptotic, necrotic and late apoptotic cells by flow cytometry. Prior to exposure, the primary cultures showed 83% cell viability. Flow cytometry following labeling of cells with a specific neuronal marker, TUJ-1, revealed 82% pure neuronal populations, whereas approximately 7% were astrocytic as shown by glial fibrillary acidic protein positivity. Exposure of primary cultures to TNF-alpha, 4-HNE, and aged Abeta1-42 gave an increased number of early apoptotic cells. We show that flow cytometry is a suitable method for quantifying effects of different stressors on neurons in primary cultures. This technique could be useful for screening and testing of pharmacological compounds relevant to neurodegenerative disorders.