Cannabinoids inhibit energetic metabolism and induce AMPK-dependent autophagy in pancreatic cancer cells.

The anti-tumoral effects of cannabinoids have been described in different tumor systems, including pancreatic adenocarcinoma, but their mechanism of action remains unclear. We used cannabinoids specific for the CB1 (ACPA) and CB2 (GW) receptors and metabolomic analyses to unravel the potential pathways mediating cannabinoid-dependent inhibition of pancreatic cancer cell growth. Panc1 cells treated with cannabinoids show elevated AMPK activation induced by a ROS-dependent increase of AMP/ATP ratio. ROS promote nuclear translocation of GAPDH, which is further amplified by AMPK, thereby attenuating glycolysis. Furthermore, ROS determine the accumulation of NADH, suggestive of a blockage in the respiratory chain, which in turn inhibits the Krebs cycle. Concomitantly, inhibition of Akt/c-Myc pathway leads to decreased activity of both the pyruvate kinase isoform M2 (PKM2), further downregulating glycolysis, and glutamine uptake. Altogether, these alterations of pancreatic cancer cell metabolism mediated by cannabinoids result in a strong induction of autophagy and in the inhibition of cell growth.

Haemoglobin glycation (Hb1Ac) increases during red blood cell storage: a MALDI-TOF mass-spectrometry-based investigation.

Haemoglobin A1c (HbA1c ) represents a key biomarker in diabetes diagnosis and management, as it is indicative of recent blood glucose concentrations. Glycation of haemoglobin is a non-enzymatic irreversible process that is promoted by the prolonged exposure of erythrocytes to high glucose concentrations, a condition that is known to occur under blood banking conditions. However, controversial data indicate no clear hint as to whether and to which extent HbA1c accumulates during red blood cell storage. Hereby, we propose the application of a validated MALDI-TOF mass-spectrometry-based method to this issue and report the observation about HbA1c levels apparently increasing over storage progression.

miR-143 regulates hexokinase 2 expression in cancer cells.

Tumor cells activate pathways that facilitate and stimulate glycolysis even in the presence of adequate levels of oxygen in order to satisfy their continuous need of molecules, such as nucleotides, ATP and fatty acids, necessary to support their rapid proliferation. Accordingly, a variety of human tumors are characterized by elevated expression levels of the hexokinase 2 isoform (HK2). Although different molecular mechanisms, including genetic and epigenetic mechanisms, have been suggested to account for the altered expression of HK2 in tumors, the potential role of microRNAs (miRNAs) in the regulation of HK2 expression has not been evaluated. Here, we report that miR-143 inhibits HK2 expression via a conserved miR-143 recognition motif located in the 3'-untranslated region (3'UTR) of HK2 mRNA. We demonstrate that miR143 inhibits HK2 expression both in primary keratinocytes and in head and neck squamous cell carcinoma (HNSCC)-derived cell lines. Importantly, we found that miR-143 inversely correlates with HK2 expression in HNSCC-derived cell lines and in primary tumors. We also report that the miRNA-dependent regulation of hexokinase expression is not limited to HK2 as miR-138 targets HK1 via a specific recognition motif located in its 3'UTR. All these data unveil a new miRNA-dependent mechanism of regulation of hexokinase expression potentially important in the regulation of glucose metabolism of cancer cells.

Red blood cell storage and cell morphology.

AIM: In this study, we performed weekly assessment of morphology-related parameters through monitoring of CPD-SAGM leuco-filtered erythrocyte concentrates from blood withdrawal until the 42nd day of storage. BACKGROUND: Liquid storage of red blood cells (RBCs) delivers a blood-derived therapeutic, which is safe, available, effective and affordable for most patients who need transfusion therapy in developed countries. However, a growing body of accumulating controversial evidences, from either biochemical or retrospective clinical studies, prompted safety concerns about longer stored RBCs. METHODS: Statistical image analysis through scanning electron microscope was coupled to osmotic fragility and erythrocyte sedimentation rate. RESULTS: We could observe that by day 21 more than 50% of RBCs displayed non-discocyte phenotypes. This observation was related to an increase in osmotic fragility, which was totally overlapped in day 0 controls and day 7 RBCs while only slightly augmented in day 14 samples. Cation dysregulation (pH internal/external alteration and potassium) might both reflect and trigger a negative feedback loop with metabolic fluxes and membrane cation pumps. CONCLUSION: Morphology parameters suggest that significant alterations to RBC morphology over storage duration occur soon after the 14th day of storage, as to become significant enough within the 21st day.

Soluble proteome investigation of cobalt effect on the carotenoidless mutant of Rhodobacter sphaeroides.

AIMS: To investigate the surviving capability of Rhodobacter sphaeroides under phototrophic conditions in the presence of high cobalt concentration and its influence on the photosynthetic apparatus biosynthesis. METHODS AND RESULTS: Cells from R. sphaeroides strain R26.1 were grown anaerobically in a medium containing 5.0 mmol l(-1) cobalt ions and in a control medium. Metal toxicity was investigated comparing the soluble proteome of Co(2+)-exposed cells and cells grown in control medium by two-dimensional gel electrophoretic analysis. Significant changes in the expression level were detected for 43 proteins, the majority (35) being up-regulated. The enzyme porphobilinogen deaminase (PBGD) was found down-regulated and its activity was investigated. CONCLUSIONS: The up-regulated enzymes mainly belong to the general category of proteins and DNA degradation enzymes, suggesting that part of the catabolic reaction products can rescue bacterial growth in photosynthetically impaired cells. Furthermore, the down-regulation of PBGD strongly indicates that this key enzyme of the tetrapyrrole and bacteriochlorophyll synthesis is directly involved in the metabolic response. SIGNIFICANCE AND IMPACT OF THE STUDY: Data and experiments show that the cobalt detrimental effect on the photosynthetic growth of R. sphaeroides is associated with an impaired expression and functioning of PBGD.

Effect of tannic acid on Lactobacillus hilgardii analysed by a proteomic approach.

AIMS: A contribution towards the elucidation of the mechanisms of tannins on bacteria growth inhibition, with particular focus on the interaction between tannins and bacterial proteins. METHODS AND RESULTS: The interaction between tannic acid (TA) and Lactobacillus hilgardii, a wine spoilage bacterium, was investigated by a combination of physiologic and proteomic approaches. Growing tests were performed on medium supplemented with TA at concentrations ranging from 100 to 1000 mg l(-1) demonstrating the inhibitory effect of TA on the growth rate. Total proteins extracted from cells unexposed and exposed to TA were then analysed by 2D-electrophoresis and significant quantitative variations with a marked decrease of protein intensity upon TA exposure were observed. Most of the proteins, identified by ESI tandem Mass Spectrometry, were metabolic enzymes of different pathways, located in cytoplasm and membrane. CONCLUSIONS: The effects of TA on cells are deduced by the involvement of metabolic enzymes, and functional proteins on the tannin-protein interaction. These results might be related to the altered functions of the cell metabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: The possible role of tannins in the inhibition of the bacterial survival and growth in a natural environment such as wine. A similar approach could be applied for evaluating the effects of tannins on food borne and pathogenic bacteria.

Identification of a furazolidone metabolite responsible for the inhibition of amino oxidases.

1 Furazolidone, a drug widely used in human and veterinary medicine, exhibits inhibition of monoamine oxidase activity, as observed in the tissues of a number of different animal species, including man. The aim of the current study was to determine which of the two possible metabolites, 3-amino-2-oxazolidone (AOZ) or beta-hydroxyethylhydrazine (HEH), a well-known carcinogenic compound, is involved in the toxicological effects reported. 2 A new spectrometric method was set up to differentiate intracellular HEH from AOZ inside cells. This method works well at low pH where both AOZ and HEH are free in solution and available to react with the chemical chromophore (DAB). 3 The results confirm that furazolidone has to be metabolized in the intact cell in order to exhibit mitochondrial monoamine oxidase inhibition, whereas AOZ itself is able to exert a reversible monoamine oxidase inhibition. AOZ also inhibits bovine serum amino oxidase. On the contrary, HEH gives irreversible inhibition of both enzymes. However, the reversible nature of the AOZ inhibition with respect to HEH suggests that the two metabolites act by different mechanisms which do not require the biotransformation of AOZ to HEH. 4 Cell lysates, previously incubated with AOZ, were directly analysed and the formation of HEH from AOZ was not detected, supporting the conclusion that the amino oxidase inhibition observed on treatment with furazolidone was attributable to AOZ and not to HEH.

Isoforms of photosystem II antenna proteins in different plant species revealed by liquid chromatography-electrospray ionization mass spectrometry.

The high selectivity offered by reversed-phase high-performance liquid chromatography on-line coupled to electrospray ionization mass spectrometry has been utilized to characterize the major and minor light-harvesting proteins of photosystem II (Lhcb). Isomeric forms of the proteins, revealed either on the basis of different hydrophobicity enabling their chromatographic separation or on the basis of different molecular masses identified within one single chromatographic peak, were readily identified in a number of monocot and dicot species. The presence of several Lhcb1 isoforms (preferably in dicots) can explain the tendency of dicot Lhcb1 to form trimeric aggregates. The Lhcb1 molecular masses ranged from 24,680 to 25,014 among different species, whereas within the same species, the isoforms differed by 14-280 mass units. All Lhcb1 proteins appear to be highly conserved among different species such that they belong to a single gene group that has several different gene family members. In all species examined, the number of isoforms corresponded more or less to the genes cloned previously. Two isoforms of Lhcb3 were found in petunia and tomato. For Lhcb6, the most divergent of all light-harvesting proteins, the greatest number of isoforms was found in petunia, tobacco, tomato, and rice. Lhcb2, Lhcb4, and Lhcb5 were present in only one form. The isoforms are assumed to play an important role in the adaptation of plants to environmental changes.

High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies.

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.

High-performance liquid chromatography coupled on-line with electrospray ionization mass spectrometry for the simultaneous separation and identification of the Synechocystis PCC 6803 phycobilisome proteins.

The complete resolution of the protein components of phycobilisome from cyanobacterium Synechocystis 6803, together with their detection and determination of molecular mass, has successfully been obtained by the combined use of HPLC coupled on-line with electrospray ionization mass spectrometry. The method proposed consists of the isolation of the light-harvesting apparatus of cyanobacterium, by simply breaking cells in low-ionic-strength buffer, and subsequent injection of the total mixture of phycobilisomes into a C4 reversed-phase column. Identification of proteins was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the samples collected from HPLC or by measuring the protein molecular mass coupling HPLC with mass spectrometry. The latter method allows the simultaneous separation of the phycobiliproteins, phycocyanin and allophycocyanin, from linker proteins and their identification, which due to their similar amino acid sequence and their similar hydrophobicity, might not be detected by denaturing SDS-PAGE. Under the experimental conditions used, the pigment phycobilin is not removed from the polypeptide backbone, determining the hydrophobicity of the phycoproteins and hence their interaction with the reversed-phase column as well as in determining the protein-protein interaction into the phycobilisome aggregation. Removal of the pigment, in fact, abolishes HPLC separation, emphasizing the essential role that the pigments play in maintaining the unusual tertiary structure of these proteins.

High performance liquid chromatography-electrospray mass spectrometry for the simultaneous resolution and identification of intrinsic thylakoid membrane proteins.

In higher plants, both photosystem I (PSI) and II (PSII) consist of membrane-embedded proteins that contain more than one transmembrane alpha helix. PSI is a multiprotein complex consisting of a core complex of thirteen proteins surrounded by four different types of light harvesting antenna proteins. Up to now, the protein components of both photosystems have been characterized by SDS-PAGE and/or immunoblotting and, therefore, identification made only on the basis of electrophoretic mobility, which is sometimes not sufficient to discriminate between individual membrane proteins. This is also complicated by the fact that some proteins, such as the antenna proteins, have almost identical molecular mass and amino acid sequence, making it difficult to identify and ascertain the relative stoichiometry of the proteins. In this paper, we report the complete resolution of the antenna proteins and most of the core components of PSI from spinach, together with the identification of proteins by molecular mass, successfully deduced by the combined use of HPLC coupled on-line with a mass spectrometer equipped with an electrospray ion source (ESI-MS). The proposed RP-HPLC-ESI-MS method holds several advantages over SDS-PAGE, including better protein separation, especially for antenna proteins, mass accuracy, speed, efficiency, and the potential to reveal isomeric forms. Moreover, the molecular masses determined by HPLC-ESI-MS are in good agreement with the molecular masses of the individual components calculated on the basis of their nucleotide-derived amino acid sequences, indicating an absence of post-translational modifications in these proteins. It follows that if the method proposed is useful for these highly hydrophobic proteins, it may be of general use for any membrane proteins, where the presence of detergent for solubilization may compromise their characterization.

Metal binding to Pseudomonas aeruginosa azurin: a kinetic investigation.

The interaction between azurin from Pseudomonas aeruginosa and Ag(I), Cu(II), Hg(II), was investigated as a function of protein state, i.e. apo-, reduced and oxidised azurin. Two different metal binding sites, characterized by two different spectroscopic absorbancies, were detected: one is accessible to Ag(I) and Cu(II) but not to Hg(II); the other one binds Ag(I) and Hg(II) but not copper. When added in stoichiometric amount, Ag(I) shows high affinity for the redox center of apo-azurin, to which it probably binds by the -SH group of Cys112; it can displace Cu(I) from reducedazurin, while it does not bind to the redox center of oxidizedazurin. Kinetic experiments show that Ag(I) binding to the reduced form is four times faster than binding to the apo-form. This result suggests that metal binding requires a conformational rearrangement of the active site of the azurin. Interaction of Ag(I) or Hg(II) ions to the second metal binding site, induces typical changes of UV spectrum and quenching of fluorescence emission.

Resolution and identification of the protein components of the photosystem II antenna system of higher plants by reversed-phase liquid chromatography with electrospray-mass spectrometric detection.

Reversed-phase liquid chromatography (RPLC) was interfaced to mass spectrometry (MS) with an electrospray ion (ESI) source for the separation and accurate molecular mass determination of the individual intrinsic membrane proteins that comprise the photosystem II (PS II) major light-harvesting complex (LHC II) and minor (CP24, CP26 and CP29) antenna system, whose molecular masses range between 22,000 and 29,000. PS II is a supramolecular complex intrinsic of the thylacoid membrane, which plays the important role in photosynthesis of capturing solar energy, and transferring it to photochemical reaction centers where energy conversion occurs. The protein components of the PS II major and minor antenna systems were extracted from spinach thylacoid membranes and separated using a butyl-silica column eluted by an acetonitrile gradient in 0.05% (v/v) aqueous trifluoroacetic acid. On-line electrospray MS allowed accurate molecular mass determination and identification of the protein components of PS II major and minor antenna system. The proposed RPLC-ESI-MS method holds several advantages over sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the conventional technique for studying membrane proteins, including a better protein separation, mass accuracy, speed and efficiency.

Effect of mercuric ions on human erythrocytes. Relationships between hypotonic swelling and cell aggregation.

This study was undertaken to verify the hypothesis that the haemolytic effect of mercuric ions on human erythrocytes is strongly decreased under swelling conditions (relative to isotonic suspensions). In fact, interaction of Hg2+ with swollen erythrocytes yields a rapid and cooperative cell aggregation, a phenomenon that appears to prevent penetration of mercuric ions into the cells and, accordingly, to avoid any haemolytic effect induced by the Hg2+ entrance. Since in vivo erythrocytes undergo big shape changes (swelling being a kind of shape modification) related to mechanical or (in some animals) osmotic stresses, the reported observations turn out to be also of some relevance for the understanding of certain toxicological effects of mercuric ions.

Role of phospholipase C and D signalling pathways in vasopressin-dependent myogenic differentiation.

Arg8-vasopressin (AVP) is a potent inducer of myogenic differentiation stimulating the expression of myogenic regulatory factors. To understand the mechanism of its effect on myogenesis, we investigated the early signals induced by AVP in myogenic target cells. In the rat skeletal muscle cell line L6, AVP selectively stimulates phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) breakdown, through the activation of phospholipases C and D (PLC, PLD), as shown by the generation of Ins(1,4,5)P3 and phosphatidylethanol (PtdEtOH), respectively. AVP induces the biphasic increase of sn-1,2-diacylglycerol (DAG) consisting in a rapid peak followed by a sustained phase, and the monophasic generation of phosphatidic acid (PA). Propranolol (a PA phosphatase inhibitor) and Zn2+ (a PLD inhibitor), abolish the sustained phase of DAG generation. Our data indicate that PtdIns-PLC activity is mainly responsible for the rapid phase of AVP-dependent DAG generation, whereas the sustained phase is dependent upon PtdCho-PLD activity and PA dephosphorylation, ruling out any significant role of DAG kinase. Modifications of PA level correlate with parallel changes of PLC activity, indicating a possible cross-talk between the two signal transduction pathways in the intact cell. PLD activation is elicited at AVP concentrations two orders of magnitude lower than those required for PLC activation. The differentiation of L6 myoblasts into multinucleated fibers is stimulated significantly by AVP at concentrations at which PLD, but not PLC, is activated. These data provide the first evidence for an important role of PLD in the mechanism of AVP-induced muscle differentiation.

Random amplified polymorphic DNA (RAPD) interpretation requires a sensitive method for the detection of amplified DNA.

The random amplified polymorphic DNA technique (RAPD) has found wide use in molecular genetics because of its speed and ease of use. For various reasons, with this method the amplified DNA fragments are produced at different concentrations between genotypes and even between polymerase chain reaction (PCR) runs. Since the detection of the multiple amplified fragments is performed routinely by agarose gel, and seldom by acrylamide gel electrophoresis, we have found that by capillary zone electrophoresis (CZE), which is more sensitive and accurate than gel electrophoresis, it is possible to unequivocally detect amplified fragments even at low concentration, avoiding polymorphism misinterpretation. CZE is also useful to make more potentially polymorphic fragments evident per random primer used, with obvious economical benefits.

Capillary electrophoresis of closely related intrinsic thylakoid membrane proteins of the photosystem II light-harvesting complex (LHC II).

The electrophoretic migration behavior of three closely related hydrophobic intrinsic membrane proteins of the photosystem II light-harvesting complex (LHC II) was investigated in free solution capillary electrophoresis at pH 8.0-10 with running electrolyte solutions containing either anionic, zwitter-ionic or nonionic detergents. The complete and repeatable separation of these proteins was accomplished with a running electrolyte solution of 25 mM Tris/192 mM glycine, pH 8.8, containing either sodium dodecyl sulfate or n-octyl beta-D-glucopyranoside at concentration up to 5.0 and 7.0 mM, respectively. Migration times and resolution of the individual LHC II intrinsic membrane proteins were sensitive to the type of detergent. The effect of detergent concentration on the electrophoretic behavior of the LHC II proteins was also investigated. Electroelution of the LHC II components separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to isolate these intrinsic membrane proteins, which were then injected onto the capillary electrophoresis system for peak identification.

Effect of mercuric ions on human erythrocytes. 1. Rate of haemolysis induced by osmotic shock as a function of incubation time.

Incubation of human erythrocytes with mercuric ions for 5 min causes a transient increase in mechanical resistance as measured by osmotic shock. After a longer incubation time (necessary for the metal to cross the cell membrane), cells recover their normal fragility; however, after 10-20 min of exposure to mercuric ions haemolysis occurs in the incubation vessel. In contrast, p-chloromercuriben-zenesulfonate (a compound known to bind mainly to band 3 of the cell membrane without passing into the cell) increases the osmotic resistance of erythrocytes, as observed transiently for mercuric ions, but does not induce haemolysis even at incubation times of 20-30 min. Therefore it seems that the interaction per se of mercuric ions with the membrane does not represent the main damaging event and therefore the toxicological effect of mercuric ions must be mostly related to subsequent processes, such as the interaction of the metal with intracytoplasmic components and/or disruption of the cytoskeleton.

Entrapment of protein protease inhibitors in red blood cells.

Aprotinin and alpha 1-proteinase inhibitor have been encapsulated in human red blood cells (RBC) by a dialysis technique that involves transient hypotonic haemolysis followed by isotonic resealing. Both protease inhibitors can be encapsulated to a considerable extent. These molecules are released only by haemolysis of the cells and that excludes the possibility of using loaded erythrocytes for a slow release of the inhibitor(s) in the blood stream. However, the stability of the two inhibitors, the evidence for the binding of aprotinin to RBC components, and the results showing inhibition of endogenous proteolytic activity indicate that the inhibitors may be valuable in blocking, at least partially, undesired intraerythrocytic proteolytic reactions.

Red blood cells as carriers for delivering of proteins.

The potential use of red blood cells as a carrier system for transport and delivery of pharmacological substances is well documented. Various methods have been attempted for encapsulation of drugs; in this review we evaluate critically all the procedures illustrating their advantages and disadvantages. Moreover, we present kinetic studies of protein encapsulation by moderate hypotonic dialysis, which allows entrapment of molecules with MW less than 50,000 Da with negligible stress of the erythrocyte membrane. Furthermore data reveal that the resealing procedure commonly used is insufficient to completely seal pores of loaded erythrocytes, allowing entrapped proteins with MW less than 12-14,000 Da to escape. However, only 20-30% of the entrapped material is released, depending on the final cytocrit, while the remaining is associated to the inner membrane or to cytosolic components. Although the method of hypotonic dialysis is known to be the one that minimally affects the biophysical and immunological properties of the red blood cell membrane, the interaction of encapsulated material with cell costituents would need to be further assessed when considering red cells as macromolecular carriers.