On the importance of anandamide structural features for its interactions with DPPC bilayers: effects on PLA2 activity

The acylethanolamide anandamide (AEA) occurs in a variety of mammalian tissues and, as a result of its action on cannabinoid receptors, exhibits several cannabimimetic activities. Moreover, some of its effects are mediated through interaction with an ion channel-type vanilloid receptor. However, the chemical features of AEA suggest that some of its biological effects could be related to physical interactions with the lipidic part of the membrane. The present work studies the effect of AEA-induced structural modifications of the dipalmitoylphosphatidylcholine (DPPC) bilayer on phospholipase A2 (PLA2) activity, which is strictly dependent on lipid bilayer features. This study, performed by 2-dimethylamino-(6-lauroyl)-naphthalene fluorescence, demonstrates that the effect of AEA on PLA2 activity is concentration-dependent. In fact, at low AEA/DPPC molar ratios (from R = 0.001 to R = 0.04), there is an increase of the enzymatic activity, which is completely inhibited for R = 0.1. X-ray diffraction data indicate that the AEA affects DPPC membrane structural properties in a concentration-dependent manner. Because the biphasic effect of increasing AEA concentrations on PLA2 activity is related to the induced modifications of membrane bilayer structural properties, we suggest that AEA-phospholipid interactions may be important to produce, at least in part, some of the similarly biphasic responses of some physiological activities to increasing concentrations of AEA.     

Ceramide triggers an NF-kappaB-dependent survival pathway through calpain

We have shown that C2 ceramide, a cell-permeable analog of this lipid second messenger, triggers an NF-kappaB dependent survival pathway that counteracts cell death. Activation of NF-kappaB and subsequent induction of prosurvival genes relies on calpain activity and is prevented on silencing of the calpain small subunit (Capn4) that is required for the function of ubiquitous calpains. We have demonstrated that p105 (NF-kappaB1) and its proteolytic product p50 can be targets of micro- and milli-calpain in vitro and that a p50 deletion mutant, lacking both the N- and the C-terminal ends, is resistant to calpain-mediated degradation. Capn4 silencing results in stabilization of endogenous p105 and p50 in diverse human cell lines. Furthermore, p105 processing and activation of NF-kappaB survival genes in response to C2 ceramide is impaired in Capn4-/- mouse embryonic fibroblasts defective in calpain activity. Altogether, these data argue for the existence of a ceramide-calpain-NF-kappaB axis with prosurvival functions.     

Macrobenthic invertebrate energy densities and ecological status in freshwater watercourses (Friuli Venezia-Giulia,Northeast Italy)

The present study provides energy density (ED) data and models for four macrobenthic invertebrate genera inhabiting freshwater lotic environments (Baetis, Hydropsyche, Rhyacophila, and Onychogomphus). Samples were collected in the hydrological freshwater network of the Region Friuli Venezia Giulia within different watercourse types (creeks, streams, rivers channels, and ditches), and energy density was directly measured using an adiabatic bomb calorimeter. Measured ED expressed in Joule g^?1 wet weight was strongly and positively correlated with percentage of dry weight (DW%) for all genera investigated (r²?>?0.9504), allowing to obtain genus-specific predictive models based on the relationship between ED and DW%. Models were validated and showed good predictive power, as 90th percentile of observed percentage errors ranged between 4.23% and 5.18% while medians ranged between 1.32% and 2.83%. ANCOVA disclosed significant differences between the models, as those for Rhyacophila and Onychogomphus differed significantly from the others. The empirical models were used to build a dataset of estimated energy density, to assess the relationship between energy density and ecological status of the monitored riverine systems, assessed in compliance with European and Italian law by the application of four different ecological indices (ICMi, RQE_IBMR, STAR_ICMi, and LIMeco). Information regarding ED levels for freshwater macrobenthic invertebrates is still neglected in biomonitoring programs, but it could be useful to interpret some ecological situations in the context of ecological status assessment, especially in relation to the trophic condition of the investigated riverine systems.

     

Pollen aroma fingerprint of two sunflower (Helianthus annuus L.) genotypes characterized by different pollen colors

Samples of fresh pollen grains, collected from capitula in full bloom from two genotypes of sunflower (Helianthus annuus L.) and characterized by a different color, i.e., white‐cream (WC) and orange (O), were analyzed by the HS‐SPME (headspace solid phase microextraction)/GC/MS technique. This study defined for the first time the fingerprint of the sunflower pollen, separated from the disc flowers, to define its contribution to the inflorescence aroma. In the GC/MS fingerprints of the WC and O genotypes, 61 and 62 volatile compounds were identified, respectively. Monoterpene hydrocarbons (34% in O vs. 28% in WC) and sesquiterpene hydrocarbons (37% in O vs. 31% in WC) were ubiquitous in all samples analyzed and represented the main chemical classes. α‐Pinene (21% in O vs. 20% in WC) and sabinene (11% in O vs. 6% in WC) were the dominant volatiles, but also a full range of aliphatic hydrocarbons and their oxygenated derivatives gave a decisive contribution to the aroma composition (10% in O vs. 12% in WC). In addition, dendrolasin (3% in O vs. 4% in WC) and some minor constituents such as (E)‐hex‐2‐en‐1‐ol (0.4% in O vs. 0.1% in WC) were pointed out not only for their contribution to the pollen scent, but also for their well‐known role in the plant ecological relationships. Having evaluated two pollen morphs with different carotenoid‐based colors, the study sought to highlight also the presence of some volatile precursors or derivatives of these pigments in the aroma. However, the pollen aroma of the two selected genotypes made a specific chemical contribution to the sunflower inflorescence scent without any influence on carotenoid derivatives.     

A comparative infrared spectroscopic study of glycoside hydrolases from extremophilic archaea revealed different molecular mechanisms of adaptation to high temperatures

The identification of the determinants of protein thermal stabilization is often pursued by comparing enzymes from hyperthermophiles with their mesophilic counterparts while direct structural comparisons among proteins and enzymes from hyperthermophiles are rather uncommon. Here, oligomeric beta-glycosidases from the hyperthermophilic archaea Sulfolobus solfataricus (Ss beta-gly), Thermosphaera aggregans (Ta beta-gly), and Pyrococcus furiosus (Pf beta-gly), have been compared. Studies of FTIR spectroscopy and kinetics of thermal inactivation showed that the three enzymes had similar secondary structure composition, but Ss beta-gly and Ta beta-gly (temperatures of melting 98.1 and 98.4 degrees C, respectively) were less stable than Pf beta-gly, which maintained its secondary structure even at 99.5 degrees C. The thermal denaturation of Pf beta-gly, followed in the presence of SDS, suggested that this enzyme is stabilized by hydrophobic interactions. A detailed inspection of the 3D-structures of these enzymes supported the experimental results: Ss beta-gly and Ta beta-gly are stabilized by a combination of ion-pairs networks and intrasubunit S-S bridges while the increased stability of Pf beta-gly resides in a more compact protein core. The different strategies of protein stabilization give experimental support to recent theories on thermophilic adaptation and suggest that different stabilization strategies could have been adopted among archaea.     

Relative quantification of membrane proteins in wild-type and prion protein (PrP)-knockout cerebellar granule neurons

Approximately 25% of eukaryotic proteins possessing homology to at least two transmembrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme α-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.     

The esterase from the thermophilic eubacterium Bacillus acidocaldarius: structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus

The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa. The enzyme, characterized as a "B-type" carboxylesterase, displays the maximal activity at 65 degrees C. Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism. We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus. In particular, the secondary structure and the thermal stability of the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fluorometry and anisotropy decays. Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus. Moreover, infrared spectra indicated a higher accessibility of the solvent ((2)H(2)O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. The data suggested an increase in the protein flexibility on increasing the temperature. Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures. Proteins 2000;40:473-481.     

A requirement for ubiquinone in ATPase activity and oxidative phosphorylation.

The suggestion that a rapidly sedimenting rough endoplasmic reticulum fraction in close association with mitochondria, is the preferred site of cytochrome P-450 synthesis has been examined. The rate of cytochrome P-450 synthesis in the different subcellular fractions has been evaluated $\text{in}\text{vivo}\text{and}\text{in}\text{vitro}$, using the immunoprecipitation technique. The results indicate that the conventional microsomal fraction (100,000 X g sediment) is the major site of cytochrome P-450 synthesis and that the rapidly sedimenting rough endoplasmic reticulum fraction associated with mitochondria is not a preferred site for the hemoprotein synthesis.     

Less Frequent and Less Severe Flu-Like Syndrome in Interferon Beta-1a Treated Multiple Sclerosis Patients with at Least One Allele Bearing the G>C Polymorphism at Position -174 of the IL-6 Promoter Gene

One of the most common adverse event of interferon beta (IFNβ) therapy for multiple sclerosis is flu-like syndrome (FLS), which has been reportedly related to increased levels of cytokines such as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Average cytokine levels can be affected by single nucleotide polymorphism in the gene promoter regions. To investigate whether IL-6 -174 G>C and TNF-α -376 G>A polymorphisms could be correlated to the incidence of FLS, and whether an anti-inflammatory/antipyretic therapy may influence FLS development, a prospective observational study was performed in 190 treatment naïve, multiple sclerosis patients who started IM IFNβ-1a 30mcg once weekly. The identification of IL-6 -174 G>C and TNF-α -376 G>A polymorphisms was achieved by performing an amplification-refractory mutation system. Serum IL-6 levels were measured using enzyme-linked immunosorbent assay in blood samples taken before therapy and then after the first and last IFNβ-1a injection of the follow-up. FLS-related symptoms were recorded by patients once per week during the first 12 weeks of therapy into a self-reported diary. We found that patients carrying at least one copy of the C allele at position -174 in the promoter of IL-6 gene produced lower levels of IL-6 and were less prone to develop FLS, which was also less severe. On the contrary, the polymorphism of TNF-α had no effect on FLS. Patients taking the first dose of anti-inflammatory/antipyretic therapy in the peri-injection period (within 1 hour) experienced a reduced FLS severity. In conclusion, the study of IL-6 -174 G>C polymorphism would allow the identification of patients lacking the C nucleotide on both alleles who are at risk of a more severe FLS, and may be addressed to a timely and stronger anti-inflammatory/antipyretic therapy for a more effective FLS prevention.