Going nuts for nuts? The trace proteome of a Cola drink, as detected via combinatorial peptide ligand libraries

The "invisible" proteome of a Cola drink, stated to be produced with a kola nut extract, has been investigated via capture with combinatorial peptide ligand libraries (CPLL). Indeed, a few proteins in the M(r) 15-20 kDa range could be identified by treating large beverage volumes (1 L) and performing the capture with CPLLs at very acidic pH values (pH 2.2) under conditions mimicking reverse-phase adsorption. Ascertaining the presence of proteins deriving from plant extracts has confirmed the genuineness of such beverage and suggests the possibility of certifying whether soft drinks present on the market are indeed made with vegetable extracts or only with artificial chemical flavoring.     

Cibacron Blue and proteomics: the mystery of the platoon missing in action

The use of Cibacron Blue columns (HiTrapBlue) in proteome analysis for removal of plasma albumin, for facilitating biomarker discovery, has not borne any fruit. In fact, the visibility of low-abundance proteins was obscured. It is here reported that, upon albumin sequestering from plasma, there is adsorption, via hydrophobic interaction, of a substantial number of plasma proteins, which are lost for subsequent analysis if the blue resin is eluted via an ion shock (2 M NaCl) or with a somewhat more robust eluant (5 M urea, 2 M thiourea, 2% CHAPS, 2% sulphobetain 3-10) as recommended by manufacturers. Such treatments, in fact, release at most 25 to 30 unique gene products, including albumin. If, however, the Affigel-Blue resin, after elution with either of the two above eluants, is further eluted with boiling 4% SDS in 25 mM DTT, all the missing proteins (amounting to at least 112 unique species) are desorbed and biomarker analysis can be conducted in a correct way. It is also suggested that such blue-resin treatment could be coupled to ProteoMiner adsorption, this coupled treatment further enhancing the chances of success for discovery of low-abundance proteins.     

Gap junction channels reconstituted in two closely apposed lipid bilayers

Intercellular communication mediated by gap junction channels plays an important role in many cellular processes. In contrast to other channels, gap junction channels span two plasma membranes resulting in an intracellular location for both ends of the junctional pore and the regulatory sites for channel gating. This configuration presents unique challenges for detailed experimental studies of junctional channel physiology and ligand-activation in situ. Availability of an appropriate model system would significantly facilitate future studies of gap junction channel function and structure. Here we show that the double-membrane channel can be reconstituted in pairs of closely apposed lipid bilayers, as experienced in cells. We have trapped the calcium-sensitive dye, arsenazo III (AIII), partially calcium-saturated (AIII-Ca), in one population of connexin32 reconstituted-liposomes, and EGTA in a second one. In such mixtures, the interaction of EGTA with AIII-Ca was measured by a large color shift from blue to red (decreased absorbance at 652 nm). The exchange of these compounds through gap junctions was proportional to these decrements. Results indicate that these connexon-mediated interliposomal channels are functional and are inhibited by the addition of alpha-glycyrrhetinic acid and by flufenamic acid, two gap junction communication inhibitors. Future use of this model system has the potential to improve our understanding of the permeability and modulation of junctional channels in its native intercellular assembly.     

Glycyrrhizin and glycyrrhetinic acid directly modulate rat cardiac performance

Root extract of liquorice is traditionally used to treat several diseases. Liquorice-derived constituents present several biological actions. In particular, glycyrrhizin and its aglycone, glycyrrhetinic acid, exhibit well-known cardiovascular properties. The aim of this research was to explore the direct cardiac activity of glycyrrhizin and glycyrrhetinic acid.The effects of synthetic glycyrrhizin and glycyrrhetinic acid were evaluated on the isolated and Langendorff perfused rat heart. The intracellular signaling involved in the effects of the two substances was analyzed on isolated and perfused heart and by Western blotting on cardiac extracts. Under basal conditions, both glycyrrhizin and glycyrrhetinic acid influenced cardiac contractility and relaxation. Glycyrrhizin induced significant positive inotropic and lusitropic effects starting from very low concentrations, while both inotropism and lusitropism were negatively affected by glycyrrhetinic acid. Both substances significantly increased heart rate. Analysis of the signal transduction mechanisms suggested that glycyrrhizin acts through the endothelin receptor type A/phospholipase C axis while glycyrrhetinic acid acts through endothelin receptor type B/Akt/nitric oxide synthase/nitric oxide axis.To our knowledge, these data reveal, for the first time, that both glycyrrhizin and glycyrrhetinic acid directly affect cardiac performance. Additional information on the physiological significance of these substances and their cardiac molecular targets may provide indication on their biomedical application.     

Identification of olive (Olea europaea) seed and pulp proteins by nLC-MS/MS via combinatorial peptide ligand libraries

Different types of extraction protocols are described for identifying proteins in seed and pulp of olive (Olea europea), by employing both conventional extraction methods and capture with ProteoMiner as well as with in house-made combinatorial peptide ligand libraries (HM-CPLLs) at pH 7.4 and at pH 2.2. Thanks to the use of CPLLs, able to dramatically amplify the signal of low-abundance species, a quite large number of compounds has been indeed identified: 61 in the seed (vs. only four reported in current literature) and 231 in the pulp (vs. 56 described so far), the deepest investigation up to the present of the olive proteome. In the seed, it highlights the presence of seed storage proteins, oleosins and histones. In the pulp, the allergenic thaumatin-like protein (Ole e 13) was confirmed, among the other 231, as the most abundant protein in the olive pulp. The present research has also been undertaken with the aim of identifying proteins in olive oil and ascertaining the relative contribution of seed and pulp proteins in their presence, if any, in oils.     

Identification and characterization of Myosin from rat testicular peritubular myoid cells

In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cells are contractile, express the cytoskeletal markers of true smooth muscle-alpha-isoactin and F-actin-and participate in the contraction of seminiferous tubules during the transport of spermatozoa and testicular fluid to the rete testis. Myosin from PMCs (PMC-myosin) was isolated from adult rat testis and purified by cycles of assembly-disassembly and sucrose gradient centrifugation. PMC-myosin was recognized by a monoclonal anti-smooth muscle myosin antibody, and the peptide sequence shared partial homology with rat smooth muscle myosin-II, MYH11 (also known as SMM-II). Most PMC-myosin (95%) was soluble in the PMC cytosol, and purified PMC-myosin did not assemble into filaments in the in vitro salt dialysis assay at 4 degrees C, but did at 20 degrees C. PMC-myosin filaments are stable to ionic strength to the same degree as gizzard MYH11 filaments, but PMC-myosin filaments were more unstable in the presence of ATP. When PMCs were induced to contract by endothelin 1, a fraction of the PMC-myosin was found to be involved in the contraction. From these results we infer that PMCs express an isoform of smooth muscle myosin-II that is characterized by solubility at physiological ionic strength, a requirement for high temperature to assemble into filaments in vitro, and instability at low ATP concentrations. PMC-myosin is part of the PMC contraction apparatus when PMCs are stimulated with endothelin 1.     

Effects of 2.45 GHz microwave fields on liposomes entrapping glycoenzyme ascorbate oxidase: evidence for oligosaccharide side chain involvement.

Previous observations reported by our group indicate that 2.45 GHz microwave fields at specific absorption rate (SAR) of 5.6 W/kg reduce the enzyme activity rate of ascorbate oxidase (AO) trapped in liposomes. In this study, we report dose-response studies on these AO containing liposomes irradiated at different SAR values (1.4, 2.8, 4.2, and 5.6 W/kg). No response was observed for SAR below 5.6 W/kg. Liposomes entrapping functional AO in its deglycated form (AO-D) were also used. In this case, no MW related enzyme activity changes were observed, demonstrating a direct involvement of oligosaccharide chains of AO. Furthermore, the catalytic properties of both AO and AO-D were not impaired by MW irradiation, neither in homogeneous solution nor loaded in liposomes, excluding possible changes in the conformation of enzyme as a mechanism. Our results suggest that the oligosaccharide chains of AO are critical to elicit the microwave observed effects on lipid membrane.     

Regulation of spermidine/spermine N1-acetyltransferase expression by cytokines and polyamines in human hepatocarcinoma cells (HepG2)

Spermidine/spermine N¹-acetyltransferase (cSAT), a key enzyme in polyamine degradation, is induced by various hepatotoxins and liver tumor promoters. In this paper we demonstrate that physiological factors, such as cytokines, control cSAT expression in HepG2 human hepatocarcinoma cells. Hepatocyte growth factor (HGF) induced the cSAT mRNA precursor (3.5 kb) at 4 h. The mature form of mRNA (1.3 kb) increased 6–8-fold between 8 and 10 h, and remained elevated until 18 h. An increase in cSAT activity (2-fold) and high levels of N¹-acetylspermidine were observed concomitantly. Interleukin-1β (IL-1β) enhanced cSAT expression (both mRNA and enzyme activity) similar to HGF, while tumor necrosis factor-α (TNFα) was less effective. This system also provides a useful means for examining the involvement of negative and positive changes of polyamines in the induction of cSAT and c-jun, a gene that participates in the control of cSAT expression. α-Difluoromethylornithine (DFMO) pretreatment, by lowering putrescine and spermidine in HGF- or IL-1β-treated cells, prevented the induction of cSAT. This effect was reversed by exogenous putrescine or spermidine. IL-1β induced c-jun mRNA more than HGF. DFMO prevented almost completely the enhancement of c-jun mRNA expression by IL-1β, and this effect was reversed by exogenous putrescine or spermidine. Therefore, we suggest that cSAT and c-jun expression is specifically regulated by polyamine-mediated mechanisms in IL-1β treated HepG2 cells. Since cSAT is inducibile by cytokines that control tumor metabolism and growth as well as tumor-host interaction, we hypothesize an involvement of cSAT in hepatoma growth. J. Cell. Physiol. 174:125–134, 1998. © 1998 Wiley-Liss, Inc.     

Comparative role of acetylation along c-SRC/ETS1 signaling pathway in bone metastatic and invasive mammary cell phenotypes

Metastatic cells switch between different modes of migration through supramolecular plasticity mechanism(s) still largely unknown. The aim of the present paper was to clarify some molecular aspects of the epigenetic control of migration of 1833-bone metastatic cells compared to MDA-MB231-parental mammary carcinoma cells. Active c-Src overexpression enhanced 1833-cell spontaneous migration and CXCR4-mediated chemoinvasion toward CXCL12 ligand. Only in metastatic cells, in fact, c-Src seemed to stabilize nuclear CXCR4-protein receptor possibly due to tyrosine phosphorylation, by impairing protein-degradative smear and causing instead an electrophoretic-mobility shift; the cytosolic steady-state level of CXCR4 was enhanced, and the protein appeared also phosphorylated. These findings suggested the triggering of unique signaling pathways in metastasis for homing of breast-cancer cells to congenial environment of specific organs. Microenvironmental stimuli activating c-Src might influence Ets1 binding to CXCR4 promoter and consequent transactivation, as well as CXCR4 post-translational regulatory mechanisms such as phosphorylation. Enhancement of Ets1 activity and CXCR4 induction by c-Src overexpression were prevented by histone deacetylase (HDAC) blockade. In contrast, HDAC inhibition with trichostatin A increased cytosolic phosphorylated CXCR4 expression in MDA-MB231 cells, but Ets1 involvement was practically unneeded. c-Src might be suggested as a bio-marker predicting metastasis sensitivity patterns to HDAC inhibitors. Rationally designed and individualized therapy may become possible as more is learned about the target molecules of HDAC's inhibitory agents and their roles, as undertaken for CXCR4 that is likely to be crucial for homing, angiogenesis and survival in a c-Src-dependent manner in bone-metastatic mammary cells.     

According to the CPLL proteome sheriffs,not all aperitifs are created equal!

Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteome of a popular aperitif in Northern Italy, called “Amaro Branzi”, stated to be an infusion of a secret herbal mixture, of which some ingredients are declared on the label, namely Angelica officinalis, Gentiana lutea and orange peel, sweetened by a final addition of honey. In order to assess the genuineness of this commercial liqueur, we have prepared extracts of the three vegetable ingredients, assessed their proteomes, and compared them to the one found in the aperitif. The amaro's proteome was identified via prior capture with CPLLs at two different pH values (2.2 and 4.8). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could confirm the presence of the following: six proteins originating from honey, 11 from orange peels, 29 from G. lutea and 46 from A. officinalis (including shared species), plus 33 species which could not be attributed to the other secret ingredients, due to paucity of genomic data on plant proteins, for a total of 93 unique gene products (merging shared proteins). This fully confirmed the genuineness of the product. Considering that most of these species could be present in trace amounts, undetectable by conventional techniques, the CPLL methodology, due to its ability to enhance the signal of trace components up to 3 to 4 orders of magnitude, could represent a powerful tool for investigating the genuineness and natural origin of commercial beverages in order to protect consumers from adulterated products.