Antioxidant action of SMe1EC2, the low-basicity derivative of the pyridoindole stobadine, in cell free chemical models and at cellular level.

The aim of the study was to evaluate the antioxidant action of SMe1EC2, the structural analogue of the hexahydropyridoindole antioxidant stobadine. The antiradical activity of SMe1EC2 was found to be higher when compared to stobadine, as determined both in cell-free model systems of AAPH-induced oxidation of dihydrorhodamine 123 and 2',7'-dichloro-dihydrofluorescein diacetate, and in the cellular system of stimulated macrophages RAW264.7. Analysis of proliferation of HUVEC and HUVEC-ST cells revealed absence of cytotoxic effect of SMe1EC2 at concentrations below 100 µM. The antioxidant activity of SMe1EC2, superior to the parent drug stobadine, is accounted for by both the higher intrinsic free radical scavenging action and by the better bioavailability of the low-basicity SMe1EC2 relative to the high-basicity stobadine.

Stability of dendriplexes formed by anti-HIV genetic material and poly(propylene imine) dendrimers in the presence of glucosaminoglycans.

There are several barriers to the application of dendriplexes formed by poly(propylene imine) dendrimers and genetic material for gene therapy. One limitation is their interaction with extracellular matrix components such as glucosaminoglycans. These can displace the genetic material from the dendriplexes, affecting their transfection activity. In this study, we analyzed the interaction between dendriplexes and the four main glucosaminoglycans (heparin, heparan sulfate, chondroitin sulfate, and hyaluronic acid) by fluorescence polarization and gel electrophoresis. Dendriplexes were formed by combining three anti-HIV antisense oligodeoxynucleotides with three poly(propylene imine) dendrimers of the fourth generation: unmodified and partially modified with maltose and maltotriose (open shell glycodendrimers). The data showed that the effect of glucosaminoglycans on dendriplexes depends on the glucosaminoglycan type and the oligosaccharide serving as the surface group of the dendrimer. Heparin at physiological concentrations destroys dendriplexes formed by open shell glycodendrimers, but dendriplexes based on unmodified poly(propylene imine) dendrimers are stable in its presence. The other glucosaminoglycans at physiological concentrations cannot destroy dendriplexes formed by any of the dendrimers studied.

Poly(propylene imine) dendrimers modified with maltose or maltotriose protect phosphorothioate oligodeoxynucleotides against nuclease activity.

The antisense oligonucleotides are promising agents for application in anti-HIV therapies. The antiretroviral nucleoside analogues administrated into circulatory system are vulnerable to nuclease degradation and require a vehicle which would not only facilitate therapeutic nucleotides into host cells, but would also provide protection against enzymatic degradation. Such potential is exhibited by poly(propylene imine) dendrimers - the branched cationic polymers easily interacting with oligonucleotides to form complexes called "dendriplexes". The aim of the present study was to evaluate the abilities of the fourth generation poly(propylene imine) dendrimers partially modified with maltose (PPI-Mal G4) or maltotriose (PPI-Mal-III G4) to protect anti-HIV antisense oligonucleotides (ODNs) from nucleolytic degradation. The ODNs (AT, GEM91, SREV) were complexed with dendrimers and subjected to cleavage by serum nucleases or endonuclease S1. The results showed that all examined dendrimers protected ODNs against nucleases contained in FBS. Both PPI-Mal G4 and PPI-Mal-III G4 dendrimers completely prevented ODNs digestion by nuclease S1 at neutral pH. The protective capabilities of investigated dendrimers were significantly weaker in acidic environment. The time stability assay showed that the dendriplexes formed by AT, GEM91, SREV and carbohydrate-modified PPI G4 dendrimers still existed after 12h incubation both in low and at neutral pH buffers. The conformational change of dendriplexes in acidic environment was proposed as possible phenomenon leading to exposition of ODNs to nuclease attack and significantly diminishing dendriplexes' resistance to nucleolitic digestion.

Hemodialysis decreases serum brain-derived neurotrophic factor concentration in humans.

In the present study we have evaluated the effect of a single hemodialysis session on the brain-derived neurotrophic factor levels in plasma [BDNF](pl) and in serum [BDNF](s) as well as on the plasma isoprostanes concentration [F(2) isoprostanes](pl), plasma total antioxidant capacity (TAC) and plasma cortisol levels in chronic kidney disease patients. Twenty male patients (age 69.8 ± 2.9 years (mean ± SE)) with end-stage renal disease undergoing maintenance hemodialysis on regular dialysis treatment for 15-71 months participated in this study. A single hemodialysis session, lasting 4.2 ± 0.1 h, resulted in a decrease (P = 0.014) in [BDNF](s) by ~42 % (2,574 ± 322 vs. 1,492 ± 327 pg ml(-1)). This was accompanied by an increase (P < 10(-4)) of [F(2)-Isoprostanes](pl) (38 ± 3 vs. 116 ± 16 pg ml(-1)), decrease (P < 10(-4)) in TAC (1,483 ± 41 vs. 983 ± 35 trolox equivalents, µmol l(-1)) and a decrease (P = 0.004) in plasma cortisol level (449.5 ± 101.2 vs. 315.3 ± 196.3 nmol l(-1)). No changes (P > 0.05) in [BDNF](pl) and the platelets count were observed after a single dialysis session. Furthermore, basal [BDNF](s) in the chronic kidney disease patients was significantly lower (P = 0.03) when compared to the age-matched control group (n = 23). We have concluded that the observed decrease in serum BDNF level after hemodialysis accompanied by elevated [F(2)-Isoprostanes](pl) and decreased plasma TAC might be caused by enhanced oxidative stress induced by hemodialysis.

Identification and analysis of the promoter region of the human DHCR24 gene: involvement of DNA methylation and histone acetylation.

Mutations in the DHCR24 gene, which encodes the cholesterol biosynthesis enzyme 3ß-hydroxysterol-∆24 reductase, result in an autosomal recessive disease called desmosterolosis. Further, reduced expression of DHCR24 is found in the temporal cortex of Alzheimer's disease patients. This suggests that variability in the regulatory regions of DHCR24 may contribute to the development of this neurodegenerative disease. In this work, we functionally characterised the proximal fragment of the human DHCR24 gene, for the first time. We show that the transcription of DHCR24 is initiated from a single CpG-rich promoter that is regulated by DNA methylation in some cell types. An activator sequence was also uncovered in the -1203/-665 bp region by reporter gene assays. Furthermore, sodium butyrate (a well-known HDAC inhibitor) increased DHCR24 expression in SH-SY5Y cells by recruiting acetylated core histones H3 and H4 to the enhancer region, as demonstrated by transient transfection and chromatin immunoprecipitation assays. Understanding the regulation of the DHCR24 gene may lead to alternative therapeutic strategies in at least some Alzheimer's patients.

An inter-laboratory validation of methods of lipid peroxidation measurement in UVA-treated human plasma samples.

Lipid peroxidation products like malondialdehyde, 4-hydroxynonenal and F(2)-isoprostanes are widely used as markers of oxidative stress in vitro and in vivo. This study reports the results of a multi-laboratory validation study by COST Action B35 to assess inter-laboratory and intra-laboratory variation in the measurement of lipid peroxidation. Human plasma samples were exposed to UVA irradiation at different doses (0, 15 J, 20 J), encoded and shipped to 15 laboratories, where analyses of malondialdehyde, 4-hydroxynonenal and isoprostanes were conducted. The results demonstrate a low within-day-variation and a good correlation of results observed on two different days. However, high coefficients of variation were observed between the laboratories. Malondialdehyde determined by HPLC was found to be the most sensitive and reproducible lipid peroxidation product in plasma upon UVA treatment. It is concluded that measurement of malondialdehyde by HPLC has good analytical validity for inter-laboratory studies on lipid peroxidation in human EDTA-plasma samples, although it is acknowledged that this may not translate to biological validity.

[Seladin-1/DHCR24: a key protein of cell homeostasis and cholesterol biosynthesis]. / Seladyna 1/DHCR24: glówne bialko homeostazy komórkowej i biosyntezy cholesterolu.

Seladin-1 is a multifunctional protein encoded by DHCR24 gene and due to its enzymatic, antioxidant,and anti-apoptotic activities, it is considered as neuroprotective agent. Seladin-1 was identified as a gene down-regulated in brain regions selectively degenerated in Alzheimer's disease. Mutations of DHCR24 gene result in inhibition of the enzymatic activity of seladin-1, causing an accumulation of desmosterol and leading to a lethal disorder called desmosterolosis. Asan enzyme of cholesterol biosynthesis, seladin-1 enhances the formation of lipid rafts and caveoles.These membrane structures are involved in the maintenance of signaling pathways and metabolic processes, such as the degradation of amyloid precursor protein, which is especially significant in the pathophysiology of Alzheimer's disease. Independently of its enzymatic activity in cholesterol biosynthesis, seladin-1 acts as a caspase-3 inhibitor, a mediator of response to oxidative and oncogenic stress, and a reactive oxygen species scavenger. However, the effects of these activities seem to be indirectly modulated by membrane cholesterol level, which in turn gives priority to seladin-1's enzymatic function in cholesterol biosynthesis, among its other functions. Seladin-1 is ubiquitously expressed, with the highest expression level in the brain and adrenal glands. Differences in seladin-1 expression profile were reported in transformed cells originating from many tissue types. Although the mechanisms of the regulation of seladin-1 activity demand further elucidation, it has already been shown that DHCR24 gene was activated byLXRa/RXRa in skin, by ERa in neurons, and by AR in prostate. Apart from estrogens and androgens,thyroid hormones, and IGF-1 also take part in the stimulation of seladin-1 expression.