Exploring interaction of beta-amyloid segment (25-35) with membrane models through paramagnetic probes.

The accumulation of beta-amyloid peptides into senile plaques is one of the hallmarks of Alzheimer's disease (AD). There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the beta-sheet oligomerization process of beta-amyloid. Abeta(25-35), the sequence of which is GSNKGAIIGLM, is a highly toxic segment of amyloid beta (Abeta)-peptides, which forms fibrillary aggregates. In the present work, two spin-labelled Abeta(25-35) analogues containing the nitroxide group of the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe at the N- or the C-terminus of the peptide sequence, respectively, were synthesized in order to investigate the peptide-membrane interaction. The orientation and associated changes of the peptide conformation in the presence of different artificial membrane models (micelles, liposomes) were evaluated by electron paramagnetic resonance and circular dichroism techniques. The results of this study allowed us to propose a model in which the C-terminal portion of the peptide is highly associated to the membrane, while the N-terminal part extends into the aqueous phase with occasional contacts with the lipid head-group region. Interestingly, the interaction of the C-terminal portion of the peptide is particularly enhanced in the presence of sodium dodecyl sulfate (SDS) molecules.     

Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene

Hypergonadotropic ovarian failure is a common cause of female infertility. It is a heterogeneous disorder that, in the most severe forms, is a result of ovarian dysgenesis (OD). Most OD cases are associated with major X-chromosome abnormalities, but the pathogenesis of this disorder is still largely undefined in patients with a normal karyotype. Animal models showed the important role in female reproduction played by the product of a gene located at Xp11.2 in humans (BMP15). BMP15 is an oocyte-specific growth/differentiation factor that stimulates folliculogenesis and granulosa cell (GC) growth. We report two sisters with a normal karyotype who are affected with hypergonadotropic ovarian failure due to OD. The familial presentation suggested a genetic origin, and candidate genes were screened for mutations. A heterozygous nonconservative substitution in the pro region of BMP15 (Y235C) was identified in both sisters but not in 210 control alleles. This mutation was inherited from the father. Mutant BMP15 appears to be processed abnormally, is associated with reduced GC growth, and antagonizes the stimulatory activity of wild-type protein on GC proliferation. In conclusion, the first natural mutation in human BMP15 is associated with familial OD, indicating that the action of BMP15 is required for the progression of human folliculogenesis. This condition represents an exceptional example of X-linked human disease exclusively affecting heterozygous females who inherited the genetic alteration from the unaffected father. BMP15 defects are involved in the pathogenesis of hypergonadotropic ovarian failure in humans.     

Proton-induced grana formation in chloroplasts. Distribution of chlorophyll-protein complexes and Photosystem II photochemistry

Lowering the pH of the incubation medium to pH 5.4 leads to grana formation morphologically similar to that induced by metal cations. The same phenomenon is observed in EDTA-washed chloroplasts, indicating that it is not due in part to electrostatic ‘masking’ by residual cations associated with the membranes. Digitonin fractionation studies have indicated that the distribution of the major chlorophyll-protein complexes between granal and stromal membrane regions is similar at pH 5.4 in the absence of Mg²⁺, and at pH 7.4 in the presence of Mg²⁺. Chlorophyll fluorescence induction studies have indicated that the primary photochemistry of Photosystem II (PS II) is stimulated by lowering the pH to 5.4, just as it is upon metal cation addition at higher pH values. The failure to observe such an increase at pH 5.4 by measuring electron transport to ferricyanide is attributed to a combination of an inhibition by this pH of electron transport at a site after Q reduction and an increase in the number of PS II centres detached from the plastoquinone pool. We conclude that the stacked configuration of chloroplast membranes leads to increased PS II primary photochemistry, which is most simply explained in terms of a redistribution of excitation energy towards PS II.     

PKA phosphorylates and inactivates AMPKα to promote efficient lipolysis

The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by β‐adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone‐sensitive lipase (HSL). TAG resynthesis is associated with high‐energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA‐mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKα1 at Ser‐173 to impede threonine (Thr‐172) phosphorylation and thus activation of AMPKα1 by LKB1 in response to lipolytic signals. Activation of AMPKα1 by LKB1 is also blocked by PKA‐mediated phosphorylation of AMPKα1 in vitro. Functional analysis of an AMPKα1 species carrying a non‐phosphorylatable mutation at Ser‐173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA‐activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.     

Biochemical characterisation of the D60A mutant of the elongation factor 1α from the archaeon Sulfolobus solfataricus

The D60A mutant of the elongation factor (EF) 1α from Sulfolobus solfataricus (Ss), was obtained as heterologous expressed protein and characterised. This substitution was carried out in order to analyse the involvement of this evolutionally conserved amino acid position in the interaction between the elongation factor and guanosine nucleotides and in the coordination of magnesium ions. The expression system used produced a folded protein able to catalyse, although to a slightly lower extent with respect to the wild-type enzyme, protein synthesis in vitro and NaCl-dependent intrinsic GTPase activity. The affinity for guanosine nucleotides was almost identical to that exhibited by wild-type SsEF-1α; vice versa, the GDP exchange rate was one order of magnitude faster on the mutated elongation factor, a property partially restored when the exchange reaction was analysed in the presence of the magnesium ions chelating agent EDTA. Finally, the D60A substitution only a little affected the high thermal stability of the elongation factor. From a structural point of view, the analysis of the data reported confirmed that this conserved carboxyl group belongs to a protein region differentiating the GDP binding mode among elongation factors from different organisms.     

New self-assembly nanoparticles and stealth liposomes for the delivery of zoledronic acid: a comparative study

Zoledronic acid (ZOL) is a drug whose potent anti-cancer activity is limited by its short plasma half-life and rapid uptake and accumulation within bone. We have recently proposed new delivery systems to avoid ZOL accumulation into the bone, thus improving extra-skeletal bioavailability. In this work, we have compared the technological and anti-cancer features of either ZOL-containing self-assembly PEGylated nanoparticles (NPs) or ZOL-encapsulating PEGylated liposomes (LIPO-ZOL). ZOL-containing NPs showed superior technological characteristics in terms of mean diameter, size distribution, and ZOL encapsulation efficiency, compared to LIPO-ZOL. Moreover, the anti-cancer activity of NPs in nude mice xenografted with prostate cancer PC3 cells was higher than that one induced by LIPO-ZOL. In addition, NPs induced the complete remission of tumour xenografts and an increase of survival time higher than that one observed with LIPO-ZOL. It has also to be considered that PC3 tumour xenografts were almost completely resistant to the anti-cancer effects induced by free ZOL. Both nanotechnological products did not induce toxic effects not affecting the mice weight nor inducing deaths. Moreover, the histological examination of some vital organs such as liver, kidney and spleen did not find any changes in terms of necrotic effects or modifications in the inflammatory infiltrate. On the other hand, NPs but not LIPO-ZOL caused a statistically significant reduction of the tumour associated macrophages (TAM) in tumour xenografts. This effect was paralleled by a significant increase of both necrotic and apoptotic indexes. The effects of the NPs were also higher in terms of neo-angiogenesis inhibition. These results suggest the future preclinical development of ZOL-encapsulating NPs in the treatment of human cancer.     

Inhibition of HIV-1 Infection by Human α-Defensin-5, a Natural Antimicrobial Peptide Expressed in the Genital and Intestinal Mucosae

Background

α-defensin-5 (HD5) is a key effector of the innate immune system with broad anti-bacterial and anti-viral activities. Specialized epithelial cells secrete HD5 in the genital and gastrointestinal mucosae, two anatomical sites that are critically involved in HIV-1 transmission and pathogenesis. We previously found that human neutrophil defensins (HNP)-1 and -2 inhibit HIV-1 entry by specific bilateral interaction both with the viral envelope and with its primary cellular receptor, CD4. Despite low amino acid identity, human defensin-5 (HD5) shares with HNPs a high degree of structural homology.

Methodology/Principal Findings

Here, we demonstrate that HD5 inhibits HIV-1 infection of primary CD4⁺ T lymphocytes at low micromolar concentration under serum-free and low-ionic-strength conditions similar to those occurring in mucosal fluids. Blockade of HIV-1 infection was observed with both primary and laboratory-adapted strains and was independent of the viral coreceptor-usage phenotype. Similar to HNPs, HD5 inhibits HIV-1 entry into the target cell by interfering with the reciprocal interaction between the external envelope glycoprotein, gp120, and CD4. At high concentrations, HD5 was also found to downmodulate expression of the CXCR4 coreceptor, but not of CCR5. Consistent with its broad spectrum of activity, antibody competition studies showed that HD5 binds to a region overlapping with the CD4- and coreceptor-binding sites of gp120, but not to the V3 loop region, which contains the major determinants of coreceptor-usage specificity.

Conclusion/Significance

These findings provide new insights into the first line of immune defense against HIV-1 at the mucosal level and open new perspectives for the development of preventive and therapeutic strategies.     

Expression of the Stilbene Synthase (StSy) Gene from Grapevine in Transgenic White Poplar Results in High Accumulation of the Antioxidant Resveratrol Glucosides

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.     

DNA decay and limited Rad53 activation after liquid holding of UV-treated nucleotide excision repair deficient S. cerevisiae cells

The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.     

Turning regenerative technologies into treatment to repair myocardial injuries

Regenerative therapies including stem cell treatments hold promise to allow curing patients affected by severe cardiac muscle diseases. However, the clinical efficacy of stem cell therapy remains elusive, so far. The two key roadblocks that still need to be overcome are the poor cell engraftment into the injured myocardium and the limited knowledge of the ideal mixture of bioactive factors to be locally delivered for restoring heart function. Thus, therapeutic strategies for cardiac repair are directed to increase the retention and functional integration of transplanted cells in the damaged myocardium or to enhance the endogenous repair mechanisms through cell-free therapies. In this context, biomaterial-based technologies and tissue engineering approaches have the potential to dramatically impact cardiac translational medicine. This review intends to offer some consideration on the cell-based and cell-free cardiac therapies, their limitations and the possible future developments.