The anti-proliferative effects of adiponectin on human lung adenocarcinoma A549 cells and oxidative stress involvement.

Adiponectin (Acrp30) plays an important role in energy metabolism and inflammation. Recently, in vivo serum Acrp30 levels have been reported to be correlated to risk of developing several types of cancers such as lung cancer, and in vitro studies have demonstrated a role for Acrp30 in the control of cell proliferation and survival. However, the molecular effects of Acrp30 on lung cancer have not yet been clearly defined. In the present study, we investigated the effects of different concentrations of Acrp30 on the A549 human alveolar epithelial cell line, an in vitro model of lung adenocarcinoma. A549 cells were exposed to various concentrations of Acrp30 and successively, proliferation, apoptosis and oxidative stress were evaluated by MTT test, caspase activity assay, flow-cytometry and western blotting analysis. Our results demonstrated that Acrp30 causes, in a time- and dose-dependent manner, a reduction of cell viability and duplication together with an increase in cell apoptosis rate. In addition, we found that Acrp30 induces an increase of lipid peroxidation evaluated by TBARS assay and a concomitant reduction of nitric oxide release, both markers of cellular oxidative stress. Taken together, our data on A549 cells provides new insight into potential involvement of Acrp30 on physio-pathologic mechanisms of lung diseases through interference with proliferation, apoptosis and oxidative status.

Role of bisphenol A as environmental factor in the promotion of non-alcoholic fatty liver disease: in vitro and clinical study.

BACKGROUND: Bisphenol A is an endocrine disrupting chemical associated with type 2 diabetes mellitus (T2DM), cardiovascular disease and liver enzyme abnormalities. AIM: To evaluate bisphenol A plasma and urine levels in non-alcoholic fatty liver disease (NAFLD) patients compared to healthy subjects. Furthermore, we evaluated, in human HepG2 cells, the effects of exposure to different concentrations of bisphenol A on both oxidative stress induction and cell proliferation. METHODS: We enrolled 60 patients with histological diagnosis of NAFLD with or without T2DM and sixty healthy subjects. In vitro, the proliferation of bisphenol A-exposed HepG2 cells at two different concentrations (0.025 and 0.05 µM) was evaluated, both at high (H-HepG2) and at low (L-HepG2) glucose concentrations for 48 h. Lipoperoxidation was assessed by thiobarbituric acid reactive substances (TBARS) assay. RESULTS: Bisphenol A levels were significantly higher in 60 NAFLD subjects, both in urine and in plasma (P < 0.0001) when compared to controls and, in this group, it appeared to be higher in 30 non-alcoholic steatohepatitis patients compared to 30 simple steatosis subjects (P < 0.05), independently from the presence of T2DM. After a bisphenol A-free diet for 1 month, NAFLD patients showed a significant reduction in bisphenol A circulating levels (P < 0.05), without a significant reduction in urine levels. H-HepG2 cells treated with bisphenol A (0.05 µM) increased proliferation compared to controls at 48 h (P < 0.0001). Bisphenol A increased TBARS levels at 48 h versus controls. CONCLUSIONS: Our study reveals a possible role of bisphenol A as an environmental factor involved in the promotion of NAFLD, particularly in T2DM patients.

Oxidative stress and ERK1/2 phosphorylation as predictors of outcome in hepatocellular carcinoma patients treated with sorafenib plus octreotide LAR.

We reported a relevant activity of the combination between sorafenib and octreotide long-acting release (LAR) in advanced hepatocellular carcinoma (HCC) patients. In this work, we have studied if oxidative stress in both serum and peripheral blood mononuclear cells (PBMC) and pERK activation status in PBMC could be predictive of response. In the 20 responsive patients, the decrease of reactive oxygen species levels was already detectable after 10 days (T10) from the beginning of sorafenib administration, and this effect was enhanced by the combined treatment with sorafenib+octreotide LAR (T21). This effect correlated with the modulation of superoxide dismutase (SOD) activity (physiological scavenger of O(2-)) and of serum nitric oxide (NO) levels. Sorafenib alone induced an increase of about 40% of NO levels and of about two-fold of SOD activity in responsive patients, and both effects were significantly potentiated by the combined treatment. We found a gradual reduction of Erk1/2 activity, as evaluated by cytofluorimetric analysis, in 15 responsive patients reaching about 50% maximal decrease at T21. On the other hand, in 17 resistant patients, Erk1/2 activity was about 80% increased at T21. The determination of both the oxidative stress status and pERK activity in PBMC has high value in the prediction of response to sorafenib+octreotide therapy in HCC patients.

Experimental study on vasoactive intestinal peptide (VIP) and its diaminopropane bound (VIP-DAP) analog in solution.

Bioactive peptides represent an exciting area of research in the fields of biochemistry and medicine and in particular the VIP/PACAP network appears to be of interest. Vasoactive intestinal peptide (VIP) is a pleiotropic factor that exerts a physiological regulatory influence and is involved in the pathogenesis of several human disorders. In this paper we have reported structural characterization of VIP by experimental and computational methods as well as a comparative analysis of the peptide with its transglutaminase catalyzed analog VIP-Diaminopropane (VIP-DAP).

Alteration in the ubiquitin structure and function in the human lens: a possible mechanism of senile cataractogenesis.

High-performance liquid chromatography purification followed by mass spectrometry analyses highlighted that human senile cataractous lens includes a 8182 Da species which is absent in the normal lens, whereas a 8566/8583 Da species is present in both lenses. Western blot analysis identified both species as ubiquitin. The species at lower molecular weight is a shorter form due to the cleavage of the C-terminal residues 73-76. As it is the last amino acid of ubiquitin which is involved in the protein degradation mechanism, we suggest that this structure modification compromises the function of ubiquitin and consequently the physiologically occurring degradation of the lens proteins.

Homology modelling of the human eukaryotic initiation factor 5A (eIF-5A).

Homology modelling of the human eIF-5A protein has been performed by using a multiple predictions strategy. As the sequence identity between the target and the template proteins is nearly 30%, which is lower than the commonly used threshold to apply with confidence the homology modelling method, we developed a specific predictive scheme by combining different sequence analyses and predictions, as well as model validation by comparison to structural experimental information. The target sequence has been used to find homologues within sequence databases and a multiple alignment has been created. Secondary structure for each single protein has been predicted and compared on the basis of the multiple sequence alignment, in order to evaluate and adjust carefully any gap. Therefore, comparative modelling has been applied to create the model of the protein on the basis of the optimized sequence alignment. The quality of the model has been checked by computational methods and the structural features have been compared to experimental information, giving us a good validation of the reliability of the model and its correspondence to the protein structure in solution. Last, the model was deposited in the Protein Data Bank to be accessible for studies on the structure-function relationships of the human eIF-5A.

Phosphorylation of seminal vesicle protein IV on Ser58 enhances its peroxidase-stimulating activity.

In this study we show that SV-IV, a major immunomodulatory, anti-inflammatory, and sperm immunoprotective protein secreted from the rat seminal vesicle epithelium, acts in vitro as a substrate of protein kinase C (PKC) competing efficiently with H1 histone, a very well known PKC substrate. Electrospray mass spectrometry (ES-MS) analysis demonstrated that approximately 10% of the native SV-IV molecules were phosphorylated by PKC and that such a modification involved only a single serine residue (Ser58) out of the 22 occurring in the protein. Interestingly, this modification produced a substantial enhancement (approximately 50%) of the native SV-IV's ability to stimulate the activity of both horseradish peroxidase (POD) and selenium-dependent glutathione peroxidase (GPX), an enzyme that is known to protect the mammalian spermatozoa from oxidative stress and loss of motility in the female genital tract following ejaculation. In contrast, the phosphorylation of SV-IV on Ser58 did not produce any effect on the anti-inflammatory properties of SV-IV, as measured by its ability to inhibit the phospholipase A2.

Synthesis of novel anti-inflammatory peptides derived from the amino-acid sequence of the bioactive protein SV-IV.

SV-IV is a basic, thermostable, secretory protein of low Mr (9758) that is synthesized by rat seminal vesicle (SV) epithelium under strict androgen transcriptional control. This protein is of obvious pharmacological interest because it has potent nonspecies-specific immunomodulatory, anti-inflammatory, and pro-coagulant activities. In evaluating the clinical relevance and the possible use in medicine of SV-IV, we became interested in the study of its structure-function relationships and aimed to identify in its polypeptide chain specific peptide fragments possessing the marked anti-inflammatory properties of the protein not associated with other biological activities (pro-coagulation and immunomodulation) typical of this molecule. By using two different experimental approaches (the fragmentation of the protein into peptide derivatives by chemical methods and the organic synthesis on solid phase of selected peptide fragments), data were obtained showing that in this protein: (a) the immunomodulatory activity is related to the structural integrity of the whole molecule; (b) the anti-inflammatory activity is located in the N-terminal region of the molecule, the 8-16 peptide fragment being the most active; (c) the identified anti-inflammatory peptide derivatives do not seem to possess pro-coagulant activity, even though this particular function has been located in the 1-70 segment of the molecule.

Inhibition of antithrombin by protein SV-IV normalizes the coagulation of hemophilic blood.

The aim of the study was to evaluate the effect of the protein Seminal Vesicle Protein No. 4 (SV-IV), a potent inhibitor of antithrombin III (antithrombin), on the coagulation of blood obtained from patients affected by hemophilia A. In the coagulating blood of these patients, the antithrombin/thrombin ratio was found to be markedly higher (about 44) than in normal individuals (about 4. 4). This high ratio was related to the low efficiency of thrombin-generating reactions induced by the factor VIII deficiency and to the high levels of free (not bound to serine proteases) antithrombin present in the hemophilic serum (antithrombin concentration was the same in normal and hemophilic plasma). The elevated concentration of free antithrombin in hemophiliacs was primarily a consequence of a reduced consumption caused by the scarce availability in the hemophilic serum of factors Xa and IIa, which are serine proteases possessing strong binding affinity for antithrombin. Addition of SV-IV to coagulating hemophilic blood reduced markedly the serum antithrombin and thrombin-antithrombin complexes, normalizing, as a consequence, the clotting time and other coagulation parameters. Similar results were obtained by using appropriate concentration of factor VIII.

The self-association of protein SV-IV and its possible functional implications.

The protein SV-IV, a major protein secreted from the rat seminal vesicle epithelium, is a basic protein with immunomodulatory, anti-inflammatory, and procoagulant activity. Predictions suggested that this protein is very flexible, with its three tyrosyl residues presumably located in water-exposed segments of the primary structure. The solution behaviour of the protein was investigated by two types of spectroscopic techniques. Modifications of the spectral characteristics of tyrosyl residues induced by changes of protein concentration were demonstrated by absorption and fluorescence experiments. In addition, secondary structure rearrangements associated with a possible self-association equilibrium were highlighted by far-UV CD spectra. The equilibrium, confirmed by chromatographic techniques, appears to control some biological properties of the protein.

Enzymatic synthesis of vasoactive intestinal peptide analogs by transglutaminase.

Vasoactive intestinal peptide is an amino acceptor and donor substrate for tissue transglutaminase (TGase) in vitro. This peptide contains a single glutamine residue, Gln16, which was identified as the amino acceptor substrate. Different gamma(glutamyl16)amine derivatives of vasoactive intestinal peptide were synthesized enzymatically in vitro. The modification is very fast when compared with that of many native substrates of TGase. The analogs 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, glycine ethyl ester and mono-dansylcadaverine of the peptide were purified by high-performance liquid chromatography on a reverse-phase column and were analyzed by electrospray mass spectrometry. When amines were absent in the assay mixture as an external amino donor, lysine residue occurring in the peptide was an effective amino donor site for TGase. Only one of the three lysine residues of vasoactive intestinal peptide, namely Lys21, was demonstrated to be involved in both inter- and intramolecular cross-link formation.

Structural organization of the human eukaryotic initiation factor 5A precursor and its site-directed variant Lys50-->Arg.

The molecular properties of the human eukaryotic initiation factor 5A precursor and its site directed Lys50-->Arg variant have been investigated and compared. Structure perturbation methods were used to gain information about the protein architecture in solution. Intrinsic and extrinsic spectroscopic probes strategically located in the protein matrix detected the independent unfolding of two molecular regions. Three cysteines out of four were titrated in the native protein and the peculiar presence of a tyrosinate band at neutral pH was detected. At alkaline pH only two tyrosines out of three were titratable in the native protein, with an apparent pK of about 9.9. Native protein and its Lys50-->Arg variant reacted in a similar fashion to guanidine and to pH variation, but differently to thermal stress. The complex thermal unfolding of both proteins indicated the presence of intermediates. Spectroscopic data showed that these intermediates are differently structured. Consequently, the two proteins seem to have different unfolding pathways.

A peptide derivative (1-70 fragment) of protein SV-IV accelerates human blood coagulation in vitro by selective inhibition of the heparin-induced antithrombin III activation process.

The effect of two peptide derivatives of the rat SV-IV (SV-IV/A: 1-70 fragment; SV-IV/B: 71-90 fragment) on human blood coagulation was investigated. The SV-IV/A fragment was found to possess the same procoagulant activity of the native protein, whereas SV-IV/B retained only a very small fraction of the activity. The results obtained strongly suggest that the procoagulant activity of SV-IV/A is due, like SV-IV, to a selective inhibition of the antithrombin III (AT III) activation process induced by heparin, an essential cofactor of AT III. The main data supporting this hypothesis are the following: 1) the concentration of active serum AT III decreases when SV-IV/A is present in the clotting system; 2) the plasma treatment with SV-IV/A reduces the concentration of AT III, but not that of other plasma serine protease inhibitors; 3) the recalcification time (RT) of the plasma treated with a rabbit anti-AT III polyclonal antiserum is not modified by SV-IV/A; 4) the presence of SV-IV/A in a reaction mixture containing pure fibrinogen, alpha-thrombin, heparin, and AT III neutralizes the thrombin inhibition induced by AT III; 5) the concentration of the thrombin-AT III complexes, occurring in sera obtained from CaCl2-coagulated plasma, is markedly reduced in the presence of SV-IV/A; 6) appropriate concentrations of heparin neutralize the inhibitory effect of either SV-IV/A or SV-IV on the AT III activity in vitro.

Structural heterogeneity, post-translational modifications, and biological activities of SV-IV, a major protein secreted from the rat seminal vesicle epithelium.

The primary structure of purified SV-IV, a major secretory protein synthesized by the rat seminal vesicle (SV) epithelium, was analysed by electrospray mass spectrometry (ES-MS). The protein was found to be highly heterogeneous. The various components were separated and identified by reversed phase high-performance liquid chromatography (HPLC) on line with ES-MS. Structural characterization of the SV-IV cyanogen bromide digests revealed the occurrence of a Val/Met substitution in about 50% of the purified protein molecules. We suggest that this mutation is the expression of a genetic polymorphism. Other minor components, corresponding to structural changes (fragmentation, deletion, and phosphorylation) of SV-IV and probably due to post-translational modifications of the native protein, were also detected. In particular, by using protein tyrosine phosphatase hydrolysis combined with ES-MS, we demonstrated that, in the phosphorylated species of SV-IV, a single phosphate group was covalently bound to the Tyr-36 residue. The significance of these findings in relation to the regulation of important biological processes, such as immune response, blood coagulation, inflammatory reaction, and mammalian reproduction, are discussed.

Water transfer energetics and solid-like packing of globular proteins.

In this article we generalize the use of a relationship based on the occurrence of some characteristic temperatures in protein unfolding, which were originally highlighted for proteins showing the unfolding enthalpy-entropy convergence. On this basis, we show how to dissect the unfolding Gibbs energy of globular proteins in terms of solid-like and liquid-like contributions, untangling the protein energetics by a scheme which does not suffer from excessive intricacy. We also provide an experimental estimate of unfavorable polar contributions to the protein stability, by which it seems possible to evaluate the number of buried residues in individual proteins. A comparison is assessed with the so-called hard sphere model of globular proteins. Results seem to reconcile the view that the protein interior is liquid-like with the observation that protein organization resembles an assembly of closely packed spheres.

Effect of galactose on alpha-crystallin.

Chromatographic separation of alpha-crystallin incubated with [3H]-labelled galactose showed the radioactivity to be concentrated in the low molecular mass subunits (20 and 40 kDa). The effect of glycation on the structural organization of alpha-crystallin was evaluated by FPLC analysis of native (pH 6.8 and 8.2) and glycated protein in dissociating conditions. Results suggest that the glycation acts on the protein surface by altering its charge distribution.

Specific interaction between cyclophilin and cyclic peptides.

Cyclophilin A, a peptidyl-prolyl cis-trans is isomerase that catalyzes the otherwise slow isomerization of Xaa-Pro imidic bond, specifically binds the immunosuppressant cyclosporin A. Herein we report evidence on binding of cyclolinopeptide A and its synthetic analogue, [Aib5,6-D-Ala8]cyclolinopeptide, to bovine cyclophilin A. Binding experiments were monitored by fluorescence, CD, and second-derivative spectroscopies, evidencing no remarkable rearrangement of protein structure organization. The possibility that cyclolinopeptide A could act as a substitute of cyclosporin A in the immunosuppression modulation is also briefly discussed.

In vitro cross-linking of calf lens alpha-crystallin by malondialdehyde.

The effect of malondialdehyde on structural features of bovine alpha-crystallin has been investigated by absorption and fluorescence spectroscopy as well as by far-UV circular dichroism. Experimental evidence suggests the occurrence of intermolecular cross-linking induced by malondialdehyde. This cross-linking does not seem to affect the tryptophan environment, as suggested by intrinsic protein fluorescence. On the contrary, the time dependence of far-UV dichroic activity indicates that the cross-linking is accompanied by a secondary structure change. The formation of high molecular mass aggregates, evidence by electrophoresis in denaturing conditions, leads to irreversible alpha-crystallin aggregation due to extensive intermolecular cross-linking. Since malondialdehyde is produced in vivo as a breakdown product of lipid peroxidation the possible involvement of this molecule in the pathological mechanism of cataract formation has been briefly discussed.

Structural organization and stability of a thermoresistant domain generated by in vivo hydrolysis of the alpha-crystallin B chain from calf lens.

A protein fragment (M(r) approximately 9000) isolated from the cortex of nonpathological calf lenses has been structurally characterized. The polypeptide structure was well organized (39% alpha-helix, 33% beta-structure, and 28% remainder) according to the far-ultraviolet circular dichroism. The fluorescence was heterogeneous for the presence of two tryptophan classes. Structure perturbation by pH and denaturant revealed cooperative structural transitions which are characteristics of a globular organization. A single-step unfolding curve induced by Gdn-HCl (midpoint = 1.38 M Gdn-HCl) was monitored by emission maximum shift as well as by far-ultraviolet circular dichroism. This transition was analyzed as a two-state process. The standard free energy of unfolding in the absence of the denaturant, delta Go (H2O), was found to be 10.80 +/- 0.25 kJ/mol at 20 degrees C and pH 7.4. The fragment also shows an unusual thermal resistance. Its structure was unperturbed up to 90 degrees C according to the fluorescence and dichroism. This last property, its peculiar amino acid composition, and the sequence of a small segment are shared, among crystallins, only with the N-terminal region of the alpha-crystallin B chain. A search for proteolysis sites along the alpha-crystallin B chain sequence revealed that it possesses specific points for proteinase attack. These sites are particularly exposed and clustered in a very flexible region in the middle of the protein sequence. They are also well represented in the C-terminal extension of the molecule while a few are buried in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular organization and structural stability of beta s-crystallin from calf lens.

beta s-Crystallin has been purified to homogeneity. Its structural features and conformational behavior have been studied in solution. Protein secondary structure was estimated by curve fitting of far-UV circular dichroism spectra, which gave 16% alpha-helix, 45% beta-sheet, 12% bends, and 27% remainders. This result indicates that the structural organization of beta s-crystallin is reasonably similar to that of other beta and gamma family members. A comparison assessed between beta s- and gamma 2-crystallin by the use of predictive methods (flexibility and volume plots) reveals that the two proteins differ in respect to their local flexibility and packing, although they show similar overall organization. The interdomain and the C-terminal regions were found to be more flexible in beta s-crystallin. This finding can be explained by the presence of smaller amino acid residues within these structural districts. The location of one out of four tryptophans, i.e., Trp-162, in a flexible and exposed region of the protein was found to be the origin of the fluorescence heterogeneity. In fact, the fluorescence emission maximum of the native protein, centered at 328 nm, is due to two emitting centers, whose emission maxima are located at 323 and 330 nm, respectively, as evidenced by acrylamide quenching of fluorescence. The effect of perturbing agents, such as pH and guanidine hydrochloride, on the conformational behavior of beta s has also been evaluated by numerous spectroscopic techniques. The range of pH stability was between 6.5 and 8.(ABSTRACT TRUNCATED AT 250 WORDS)