Search

In this study, we explored the ability of Annurca apple flesh polyphenol extract (AFPE) to affect the activity of key enzymes involved in neurodegenerative disorders&mdash, in particular, Acetyl- and Butirryl-cholinesterases, and type A and B monoamine oxidase. The effect of AFPE on enzyme activity was analyzed by in vitro enzyme assays, and the results showed concentration-dependent enzyme inhibition, with IC50 values corresponding to 859 &plusmn, 18 &micro, M and 966 &plusmn, 72 &micro, M for AChE and BuChE respectively, and IC50 corresponding to 145 &plusmn, 3 &micro, M and 199 &plusmn, 7 &micro, M for MAO-A and MAO-B, respectively, with a preference for MAO-A. Moreover, in this concentration range, AFPE did not affect the viability of human neuroblastoma SH-SY5Y and fibroblast BJ-5ta cell lines, as determined by an MTT assay. In conclusion, our results demonstrate that AFPE shows the new biological properties of inhibiting the activity of enzymes that are involved in brain functions, neurodegenerative disorders, and aging.

Among matrix metalloproteinases (MMPs), MMP-9/2 are key enzymes involved in the proteolysis of extracellular matrices in the inflammatory process and in cancer. Since MMP-9/2 expression levels, activity, and secretion is up-regulated during inflammation in response to pro-inflammatory cytokines, such as interleukin-6 (IL-6), many efforts have been devoted to identifying factors that could inhibit the IL-6-induced MMP-9/2 expression. Up to now, several reports indicated that polyphenols from fruits and vegetables are among the major components of health promotion for their antioxidant properties and also for their anti-inflammatory and anti-cancer agents. Among plant derived polyphenols, lemon (Citrus limon) peel extract (LPE) shows anti-cancer properties in various cancer types. In our previous work, we demonstrated that LPE can reduce IL-6-induced migration/invasiveness and MMP-9/2 up-regulation in some gastric cancer cell lines. This study aims to exploit the anti-cancer properties of LPE using an in vitro system model of inflammation, consisting of IL-6-exposed human primary colon cancer cells. We first analyzed the effect of LPE on IL-6-induced cell migration and invasiveness by wound healing and Boyden chamber assay, respectively. The MMP-2 mRNA expression levels and gelatinolytic activity in the cell culture media were determined by q-PCR analysis and gelatin zymography, respectively, and finally, the effects of LPE on IL-6-induced JAK2/STAT3 signaling pathways have been investigated by Western blotting analysis. Our results show that LPE is able to inhibit the IL-6-dependent cell migration and invasiveness associated with the up-regulation of MMP-2 expression levels and that these effects are correlated to the STAT3 phosphorylation in human primary T88 and T93 colon cancer cells.

Mannose-binding lectin (MBL)-Associated Serine Proteases (MASP)-1 and 3, key enzymes in the lectin complement pathway of innate immune response, are also expressed in glioma cell lines. We investigated MASP-1 and MASP-3 expression during dibutyryl cyclic AMP (dbcAMP)- or Interleukin-6 (rIL-6)-induced astrocytic differentiation of C6 glioma cells. Our results demonstrate that C6 cells express basal levels of MASP-1 and MASP-3 and following exposure to dbcAMP or IL-6, a consistent MASP-1 and MASP-3 mRNA up-regulation was found, with a behavior similar to that showed by the fibrillary acidic protein (GFAP). Furthermore, in cell conditioned media, rIL-6 stimulated MASP-3 secretion which reached levels similar to those obtained by dbcAMP treatment. Moreover, the detection of a 46-kDa MASP-3 suggested its processing to the mature form in the extracellular cell medium. Interestingly, the H89 PKA inhibitor, mostly affected dbcAMP-induced MASP-1 and MASP-3 mRNA levels, compared to that of rIL-6, suggesting that cAMP/PKA pathway contributes to MASP-1 and MASP-3 up-regulation. MASP-1 and MASP-3 expression increase was concomitant with dbcAMP- or rIL-6-induced phosphorylation of STAT3. Our findings suggest that the increase in intracellular cAMP concentration or rIL-6 stimulation can play a role in innate immunity enhancing MASP-1 and MASP-3 expression level in C6 glioma cells.

Skeletal muscle is an extremely plastic tissue for its ability to respond to different stimuli such as physiological variation and external stress. Muscle hypertrophy involves an increase in muscle mass, changes in myofibril composition and adaptation of metabolic pathways. Plasticity of skeletal muscle in response to exercise training is also caused by proliferation and differentiation of the satellite muscle stem cells in response to various growth and differentiation factors. This process is mainly mediated by myokines secreted during skeletal muscle contraction. Myokines are proteins that act as hormones both locally in the muscle and/or in an endocrine manner in other organs, mainly liver, brain and adipose tissue. Myostatin, known as growth differentiation factor-8, a myokine member of transforming growth factor-b (TGF-b) superfamily, can act on muscle cells in an autocrine manner leading to inhibition of muscle myogenesis. Muscle myostatin expression and its plasma concentration are downregulated after acute and long-term physical exercise thus allowing muscle hypertrophy. In addition, myostatin is correlated to obesity and insulin resistance for its ability to affect energy metabolism and insulin-sensitivity in muscle cells, respectively. These findings reveal that myostatin may have potential therapeutic applications to treat muscle atrophy diseases in humans. Even in sports, drugs able to inhibit myostatin expression can lead athletes to increase their sport performance. Here, we present a brief overview of myostatin and its role in biological mechanisms involved in exercise-induced plasticity of skeletal muscle.

Human health implies complex mechanisms and involves diseases prevention aimed at ensuring a psycho-physical wellbeing homoeostasis. The search for longevity can be related to the slowdown in aging, and psycho-physical wellbeing is often related to the lifestyle, mainly to the human physical activity and nutrition. Circadian rhythms are processes that affect the behaviour, physiology and metabolism of mammals across cyclic periods of 24 hours. These rhythms are regulated by multiple physiological systems, whose key elements are the alternation between light and dark and between food consumption and periods of fasting. Therefore, during evolution, a constant adaptation to natural rhythms by humans has been established towards the surrounding environment determining periods of food consumption and periods of fasting coinciding with rest. Recent sociological surveys have shown that there is a slight increase in the number of people who devote themselves to a constant physical/sport activity, but this is an ephemeral percentage compared to the dramatic increase in the incidence of diseases related to aging such as obesity and diabetes, easily preventable with healthy lifestyles. Therefore, the connection between circadian rhythms, physical activity and lifestyles represents an important feature involved in human longevity. Here, a survive of the biological mechanisms, implied in this behaviour, is presented.

Among plant polyphenols, lemon peels extract (LPE) from the residues of the industrial processing of lemon (Citrus limon) shows anti-proliferative properties in cancer cells and anticholinesterase activity. In this study, we analyze the anti-cancer properties of LPE on migration and invasiveness in MKN-28 and AGS human gastric cancer cell lines either in the absence or presence of the pro-inflammatory cytokine IL-6. We find that the pretreatment with non-cytotoxic concentrations (0.5&ndash, 1 &mu, g/ml of gallic acid equivalent) of LPE inhibits interleukin-6 (IL-6)-induced cell migration and invasiveness in MKN-28 and AGS cells, as analyzed by wound and matrigel assays. Pretreatment with LPE is able to prevent either IL-6-induced matrix metalloproteinases (MMP)-9/2 activity, as assessed by gel zymography, or mRNA and protein MMP-9/2 expression, as evaluated by qPCR and Western blotting analysis, respectively. These LPE effects are associated with an IL-6-dependent STAT3 signaling pathway in MKN-28 and AGS cells. Furthermore, LPE shows acetylcholinesterase inhibitory activity when assayed by the Ellman method. In conclusion, our results demonstrate that LPE reduces the invasiveness of gastric MKN-28 and AGS cancer cells through the reduction of IL-6-induced MMP-9/2 up-regulation. Therefore, these data suggest that LPE exerts a protective role against the metastatic process in gastric cancer.

Multitargeting or polypharmacological approaches, looking for single chemical entities retaining the ability to bind two or more molecular targets, are a potentially powerful strategy to fight complex, multifactorial pathologies. Unfortunately, the search for multiligand agents is challenging because only a small subset of molecules contained in molecular databases are bioactive and even fewer are active on a preselected set of multiple targets. However, collections of natural compounds feature a significantly higher fraction of bioactive molecules than synthetic ones. In this view, we searched our library of 1175 natural compounds from marine sources for molecules including a 2-aminoimidazole+aromatic group motif, found in known compounds active on single relevant targets for Alzheimer's disease (AD). This identified two molecules, a pseudozoanthoxanthin (1) and a bromo-pyrrole alkaloid (2), which were predicted by a computational approach to possess interesting multitarget profiles on AD target proteins. Biochemical assays experimentally confirmed their biological activities. The two compounds inhibit acetylcholinesterase, butyrylcholinesterase, and beta-secretase enzymes in high-to submicromolar range. They are also able to prevent and revert beta-amyloid (A beta) aggregation of both A beta(1-40) and A beta(1-42) peptides, with 1 being more active than 2. Preliminary in vivo studies suggest that compound 1 is able to restore cholinergic corticohippocampal functional connectivity.

An ecofriendly synthetic pathway for the synthesis of donepezil precursors is described. Alternative energy sources were used for the total synthesis in order to improve yields, regioselectively, and rate of each synthetic step and to reduce the coproduction of waste at the same time. For all products, characterized by an improved structural rigidity respect to donepezil, the inhibitor activity on AChE, the selectivity vs BuChE, the side-activity on BACE-1, and the effect on SHSY-5Y neuroblastoma cells viability were tested. Two potential new lead compounds for a dual therapeutic strategy against Alzheimer’s disease were envisaged.

In this manuscript we report an integrated study to develop simple choline esters as cholinergic agents potentially useful against the Alzheimer disease. In previously reported experiments we demonstrated the capability of the pivaloylcholine to exert cholinergic effects into the Central Nervous System, so we decided to explore small variants of choline esters. The knowledge of crystallographic models of the enzymes involved in the hydrolysis of the acetylcholine allowed to consider the steric compatibility of some choline derivatives within their catalytic sites. The purpose of the work was to find out analogues with increased selectivity toward the acetylcholinesterase versus the butyrrylcholinesterase. Theoretical models were compared to enzymatic tests carried out with both enzymes and two different methods. In this screening we have selected two candidates for the in vivo experiments with pre-treated rats. Their electroencephalographic profiles were recorded and averaged before and after the intraperitoneal treatment with two compounds in comparison to the pivaloyl lead ester. The results demonstrated that one of the esters can exert biological effects similar to the parent compound.

Protease Nexin-1, a 43-kDa glycoprotein, is a major physiological thrombin inhibitor involved in the modulation of nerve cell plasticity. Recombinant rat Protease Nexin-1 (rPN-1) was efficiently produced in Escherichia coli using a T7 RNA polymerase based expression system and purified by heparin-sepharose affinity chromatography yielding 3 mg of protein per liter of cell culture. The purity and chemical identity of rPN-1 were assessed by SDS-PAGE, Reverse Phase- High Performance Liquid Chromatography, mass spectrometry and two-dimensional-gel electrophoresis. Conformational analysis by circular dichroism and fluorescence spectroscopy revealed the presence of mixed α/β secondary structure and the prevailing localization of Trp-residues in rather polar environments. Fluorescence titration of rPN-1 with heparin indicated that rPN-1 binds heparin with high affinity. Furthermore, the formation of a SDS-stable 1:1 thrombin–rPN-1 complex, monitored by SDS-PAGE, confirmed the native-like structure of rPN-1. Finally, the cellular effects of rPN-1, such as its ability to promote neurite outgrowth in neuroblastoma cells, were found to be very similar to those elicited by natural PN-1. Altogether, our results demonstrate that glycosylation does not alter neither structure nor function of PN-1 and that E. coli is a suitable expression system for obtaining milligram quantities of pure and fully active rPN-1 for structural and functional studies.

The interaction between 2',3'-cyclic nucleotide 3'-phosphodiesterase and guanine/adenine nucleotides was investigated. The binding of purine nucleotides to 2',3'-cyclic nucleotide 3'-phosphodiesterase was revealed by both direct and indirect methods. In fact, surface plasmon resonance experiments, triphosphatase activity measurements, and fluorescence experiments revealed that 2',3'-cyclic nucleotide 3'-phosphodiesterase binds purine nucleotide triphosphates with an affinity higher than that displayed for diphosphates; on the contrary, the affinity for both purine monophosphates and pyrimidine nucleotides was negligible. An interpretation of biological experimental data was achieved by a building of 2',3'-cyclic nucleotide 3'-phosphodiesterase N-terminal molecular model. The structural elements responsible for nucleotide binding were identified and potential complexes between the N-terminal domain of CNP-ase and nucleotide were analyzed by docking simulations. Therefore, our findings suggest new functional and structural property of the N-terminal domain of CNPase.

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. Thrombin selectively cleaves PAR1, PAR3, and PAR4 to induce activation of platelets and vascular cells, while PAR2 is preferentially cleaved by trypsin. In pathological situations, other proteolytic enzymes may be generated in the circulation and could modify the responses of PARs by cleaving their extracellular domains. To assess the ability of such proteases to activate or inactivate PARs, we designed a strategy for locating cleavage sites on the exofacial NH(2)-terminal fragments of the receptors. The first extracellular segments of PAR1 (PAR1E) and PAR2 (PAR2E) expressed as recombinant proteins in Escherichia coli were incubated with a series of proteases likely to be encountered in the circulation during thrombosis or inflammation. Kinetic and dose-response studies were performed, and the cleavage products were analyzed by MALDI-TOF mass spectrometry. Thrombin cleaved PAR1E at the Arg41-Ser42 activation site at concentrations known to induce cellular activation, supporting a native conformation of the recombinant polypeptide. Plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3 cleaved at multiple sites and would be expected to disable PAR1 by cleaving COOH-terminal to the activation site. Cleavage specificities were further confirmed using activation site defective PAR1E S42P mutant polypeptides. Surface plasmon resonance studies on immobilized PAR1E or PAR1E S42P were consistent with cleavage results obtained in solution and allowed us to determine affinities of PAR1E-thrombin binding. FACS analyses of intact platelets confirmed the cleavage of PAR1 downstream of the Arg41-Ser42 site. Mass spectrometry studies of PAR2E predicted activation of PAR2 by trypsin through cleavage at the Arg36-Ser37 site, no effect of thrombin, and inactivation of the receptor by plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3. The inhibitory effect of elastase was confirmed on native PAR1 and PAR2 on the basis of Ca(2+) signaling studies in endothelial cells. It was concluded that none of the main proteases generated during fibrinolysis or inflammation appears to be able to signal through PAR1 or PAR2. This strategy provides results which can be extended to the native receptor to predict its activation or inactivation, and it could likewise be used to study other PARs or protease-dependent processes.

In the central nervous system, alteration of glial cell differentiation can affect brain functions. Polychlorinated biphenyls (PCBs) are persistent environmental chemical contaminants that exert neurotoxic effects in glial and neuronal cells. We examined the effects of a commercial mixture of PCBs, Aroclor1254 (A1254) on astrocytic differentiation of glial cells, using the rat C6 cell line as in vitro model. The exposure for 24 h to sub-toxic concentrations of A1254 (3 or 9 μM) impaired dibutyryl cAMP-induced astrocytic differentiation as showed by the decrease of glial fibrillary acidic protein (GFAP) protein levels and inhibition in change of cell morphology toward an astrocytic phenotype. The A1254 inhibition was restored by the addition of a protein kinase C (PKC) inhibitor, bisindolylmaleimide (bis), therefore indicating that PCBs disturbed the cAMP-induced astrocytic differentiation of C6 cells via the PKC pathway. The phosphorylation of signal transducer and activator of transcription 3 (STAT3) is essential for cAMP-induced transcription of GFAP promoter in C6 cells. Our results indicated that the exposure to A1254 (3 or 9 μM) for 24 h suppressed cAMP-induced STAT3 phosphorylation. Moreover, A1254 reduced cAMP-dependent phosphorylation of STAT3 requires inhibition of PKC activity. Together, our results suggest that PCBs induce perturbation in cAMP/PKA and PKC signaling pathway during astrocytic differentiation of glial cells., Highlights • A1254, a polychlorinated biphenyls mixture, exerts neurotoxic effects in C6 glial cells. • Sub-toxic concentrations of A1254 decrease levels of glial fibrillary acidic protein. • Sub-toxic concentrations of A1254 inhibit cAMP-induced astrocytic differentiation. • A1254 reduces cAMP-dependent phosphorylation of STAT3 on Ser727 via protein kinase C. • Polychlorinated biphenyls may affect astrocytic differentiation of glial cells.

The D60A mutant of the elongation factor (EF) 1alpha 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-1alpha; 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.

Human interleukin-6 or B-cell stimulatory factor-2 is a cytokine involved in acute phase and immune response. Cloning of cDNA for human interleukin-6 in the pT7.7 expression plasmid under the control of a bacteriophage T7 RNA polymerase promoter system allows rapid production of the cytokine in Escherichia coli. Upon cell induction with isopropyl thiogalactopyranoside, recombinant human interleukin-6 is overexpressed and forms insoluble inclusion bodies. Solubilization of the protein with 6 M guanidine hydrochloride and refolding in the presence of a reduction/oxidation system results in a quantitative recovery of recombinant human interleukin-6. This material is already 70% pure and can be further purified to homogeneity with a single passage over a weak anionic-exchange column. Extended structural characterization of the purified protein by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry demonstrates that recombinant human interleukin-6 is identical to the natural protein both in amino acid sequence and S-S bridge content. However, it contains a minor component accounting for about 20% of the entire translated protein which exhibits a Met-Ala dipeptide extension at the N-terminus. Purified recombinant human interleukin-6 is biologically active because it is able to induce at least 70-fold the human C-reactive promoter transfected in human hepatoma Hep 3B cells and is stable for several months in 10% glycerol at 4 degrees C. The expression system described in the present work has the main advantage of producing a high yield of recombinant human interleukin-6 (about 25 mg/l) combined with a very simple purification scheme.

The interaction between 2',3'-cyclic nucleotide 3'-phosphodiesterase and guanine/adenine nucleotides was investigated. The binding of purine nucleotides to 2',3'-cyclic nucleotide 3'-phosphodiesterase was revealed by both direct and indirect methods. In fact, surface plasmon resonance experiments, triphosphatase activity measurements, and fluorescence experiments revealed that 2',3'-cyclic nucleotide 3'-phosphodiesterase binds purine nucleotide triphosphates with an affinity higher than that displayed for diphosphates; on the contrary, the affinity for both purine monophosphates and pyrimidine nucleotides was negligible. An interpretation of biological experimental data was achieved by a building of 2',3'-cyclic nucleotide 3'-phosphodiesterase N-terminal molecular model. The structural elements responsible for nucleotide binding were identified and potential complexes between the N-terminal domain of CNP-ase and nucleotide were analyzed by docking simulations. Therefore, our findings suggest new functional and structural property of the N-terminal domain of CNPase.

The screening of a differential library from precursor and differentiated oligodendrocytes, obtained through the representational difference analysis (RDA) technique, has generated a number of cDNA recombinants corresponding to mRNA coding for known and unknown proteins: (1) mRNA coding for proteins involved in protein synthesis, (2) mRNA coding for proteins involved in the organization of the cytoskeleton, and (3) mRNA coding for proteins of unknown function. The expression profile of the mRNA was studied by Northern blot hybridization to the poly-A+ mRNA from primary rat progenitor and differentiated oligodendrocytes. In most cases, hybridization to the precursor was higher than hybridization to the differentiated mRNA, supporting the validity of the differential screening. Hybridization of the cDNA to rat cerebral hemisphere and brain stem poly-A+ mRNA, isolated from 1- to 90-day-old rats, confirms the results obtained with the mRNA from differentiating oligodendrocytes. The intensity of the hybridization bands decreases as differentiation proceeds. The pattern of expression observed in oligodendrocytes is different from that found in the brain only in the case of the nexin-1 mRNA, the level of which remains essentially constant throughout differentiation both in the brain stem and in the cerebral hemispheres, in agreement with the published data. In contrast, the intensity of hybridization to the oligodendrocyte mRNA is dramatically lower in the differentiated cells compared with the progenitor oligodendrocyte cells. Some of the recombinant cDNA represent mRNA sequences present at high frequency distribution in the cells, while others belong to the rare sequences group. Six recombinants code for proteins of the ribosomal family, suggesting that of approximately 70 known ribosomal proteins, only a few are upregulated during oligodendrocyte differentiation. The third category of open reading frame (ORF) is represented by rare messengers coding for proteins of unknown functions and includes six clones: RDA 279, 11, 95, 96, 254, and 288. GLIA 39:114–123, 2002. © 2002 Wiley-Liss, Inc.

Lipocortin or annexin 1 is a calcium-dependent phospholipid-binding protein which probably acts as a glucocorticoid- regulated anti-inflammatory factor. cDNA for human lipocortin 1 was cloned in the pT7.7 expression plasmid under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl thio-beta-D-galactoside, large amounts of the protein were produced and accumulated in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 30 mg/l bacterial culture. Electrospray mass spectrometric analysis of the purified protein demonstrated that the recombinant product corresponds to the native human lipocortin 1, without the initial methionine and with a free N-terminal alanine; tryptic peptide mapping by fast-atom-bombardment mass spectrometry showed that the recombinant protein contains cysteine residues at positions 263 and 324 with free thiol groups, whereas Cys270 and Cys343 are probably involved in an intrachain disulfide bridge. Recombinant human lipocortin 1 reduces the carrageenin-induced paw oedema in rat in vivo and inhibits porcine pancreatic phospholipase A2 activity in vitro; in both cases, a dose-related response is observed.

Three internal-amino acid deletions of amino acids 171-179 of human interleukin 6 (IL-6) were introduced at the cDNA level. While all deletion proteins were biologically inactive, immunoprecipitations with a set of conformation-specific anti-(IL-6) monoclonal antibodies showed that only mutant delta 177-179 does not present major alterations in folding. This finding, together with the observation that delta 177-179 is not able to compete with IL-6 for binding to the soluble human IL-6 receptor, suggested that some or all of these three residues participate to the composition of the receptor-binding site of human IL-6. A large number of single-amino-acid-substitution mutants were generated in residues 177, 178 and 179. Their detailed analysis revealed that Arg179 is crucial for activity in mouse cells, because all amino acid substitutions in this position cause a dramatic drop of biological activity on murine hybridoma cells without affecting the overall protein folding. The only substitution which preserved some residual activity was the conservative Arg to Lys change. This demonstrates the absolute requirement for a positive charge in position 179 for the interaction of human IL-6 with its receptor., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

By cDNA mutagenesis, we have constructed internal and C-terminal deletions (delta 21-51, delta 52-97, delta 97-104, delta 127-174, delta 97-184 and delta 134-184) in human interleukin-6 (hIL-6). All those deletion-carrying hIL-6 (delta hIL-6) proteins were then produced in Xenopus laevis oocytes and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that, at least in frog oocytes, the first potential N-glycosylation site (Asn45) is utilized exclusively. The IL-6 conformation of these deletion-carrying proteins has been studied by immunoprecipitation with two kinds of monoclonal antibodies (mAb's): mAb's that show preference towards denatured hIL-6, or conformation-specific mAb's. The binding pattern of these two series of mAb's indicated that the IL-6 conformation has been largely destroyed for four of our delta-proteins. Proteins delta 21-51 and delta 127-174 have kept a part of the IL-6 tertiary structure since they are still recognized by some conformation-specific mAb's. All of these delta hIL-6 proteins were inactive in the IL-6 hybridoma growth factor (HGF) assay and unable to inhibit the HGF activity of the recombinant human wild-type IL-6 (wt hIL-6). Moreover, the oocyte-synthesized delta hIL-6 (delta 21-51, delta 127-174, delta 97-184, delta 134-184) did not bind to the IL-6 receptor. Finally, we have produced two proteins with aa 29-33 or 97-104 substituted by corresponding murine IL-6 (mIL-6) sequences.(ABSTRACT TRUNCATED AT 250 WORDS), Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Transcription of the human C-reactive protein (CRP) gene is induced by interleukin-6 (IL-6) during acute inflammation. Important information for inducible CRP expression is located within the 90 bases preceding the transcriptional start site. We show that the CRP promoter contains two adjacent binding sites (beta and alpha) that interact with at least two hepatocyte-specific nuclear proteins, H-APF-1 and H-APF-2. Point mutations that abolish or reduce binding drastically affect the level of CRP gene expression. Binding to beta is identical when extracts from uninduced or IL-6-induced Hep3B cells are used. On the contrary, both quantitative and qualitative changes in the alpha binding can be detected with extracts from uninduced cells or from cells treated with IL-6 or IL-6 + cycloheximide. A synthetic promoter based on the multimerization of the beta-binding domain, but not of the alpha-domain, is highly inducible when transfected in hepatoma cells. These results are discussed in relation to the structure of the promoter region of other acute phase inducible genes.

Human C-reactive protein (CRP) is the major acute phase reactant during acute inflammation. The human CRP promoter is expressed in an inducible and cell-specific manner when linked to the bacterial CAT gene and transfected into human hepatoma cell cultures. In this paper we analyze the effect of several recombinant cytokines or CRP promoter inducibility in human Hep3B cells. When cytokines are tested singly the major inducer of CRP-CAT fusions is interleukin-6 (IL-6). Maximal CAT gene expression, however, is only achieved when both interleukin-1 beta (IL-1 beta) and IL-6 are present. The response to the two cytokines is cooperative. Cooperativity is maintained when the CRP promoter is linked to a different coding region, that of the bacterial neomycin phosphotransferase II gene. With a series of 5' and 3' deletions we show the existence of two distinct and independent regions responsive to IL-6 and located upstream to the TATA box. The IL-1 effect is exerted at the level of downstream sequences that are probably important for optimal mRNA translatability or nuclear-cytoplasmic transport. Inducibility is not influenced by the activation of protein kinases C or A and does not require new protein synthesis.

Several studies demonstrated a correlation between green tea consumption and a reduced cancer risk. Among different components, green tea polyphenols have been identified as molecules responsible for the beneficial effects showed by the green tea against oxidative stress and cell invasiveness. In this study, we investigated the effects of green tea polyphenol extracts (GTPs) in human gastric MKN-28 cell line. To this aim, we have first evaluated the effect of GTPs on oxidative stress induced cell injury. The pre-treatment with 10−4 M catechin equivalents of GTPs exerts a protective effect on xanthine–xanthine oxidase induced cell cytotoxicity, thus confirming the anti-oxidant properties of GTPs. The effect of GTPs was also extended to the invasive ability of MKN-28 cells stimulated with TNF-α or LPS, as pro-inflammatory factors. Migration and matrigel invasion assays demonstrated that GTPs exposure (10−6 M) prevents the increase in cell invasiveness induced by TNF-α or LPS. Finally, we have analyzed the effect of GTPs on the levels of Matrix Metalloproteinases (MMP)-9/2, whose expression is up-regulated by TNF-α or LPS. Our results indicated that the pre-treatment with GTPs was able to reduce MMP-9/2 expression at both protein and enzyme activity levels in the conditioned media of TNF-α or LPS stimulated MKN-28 cells. In conclusion, our results demonstrated that green tea polyphenol extract reduces the invasiveness of gastric MKN-28 cancer cells through the reduction of TNF-α or LPS induced MMP-9/2 up-regulation. Therefore, these data support the hypothesis that GTPs could exert a protective role against the metastatic process in gastric cancer., Graphical abstract, Highlights • Green tea polyphenols prevent oxidative stress induced cytotoxicity in MKN-28 cells. • Green tea polyphenols reduce invasiveness of MKN-28 cells. • Green tea polyphenols reduce MMP-9/2 levels induced by TNF-alpha or LPS. • Green tea polyphenols might prevent cell-invasiveness during inflammation.

Protease Nexin-1 (PN-1) or Serpine2 is a physiological regulator of extracellular proteases as thrombin and urokinase (uPA) in the brain. Besides, PN-1 is also implicated in some human cancers and further identified as a substrate for Matrix Metalloproteinase (MMP)-9, a key enzyme in tumor invasiveness. Our aim was to study the role of PN-1 in the migration and invasive potential of glioma cells, using the rat C6 glioma cell line as stable clones transfected with pAVU6+27 vector expressing PN-1 short-hairpin RNA. We find that PN-1 knockdown enhanced the in vitro migration and invasiveness of C6 cells which also showed a strong gelatinolytic activity by in situ zymography. PN-1 silencing did not alter prothrombin whereas increased uPA, MMP-9 and MMP-2 expression levels and gelatinolytic activity in a conditioned medium from stable C6 cells. Selective inhibitors for MMP-9 (Inhibitor I), MMP-2 (Inhibitor III) or exogenous recombinant PN-1 added to the culture medium of C6 silenced cells restored either the migration and invasive ability or gelatinolytic activity thus validating the specificity of PN-1 silencing strategy. Phosphorylation levels of extracellular signal-related kinases (Erk1/2 and p38 MAPK) involved in MMP-9 and MMP-2 signaling were increased in PN-1 silenced cells. This study shows that PN-1 affects glioma cell migration and invasiveness through the regulation of uPA and MMP-9/2 expression levels which contribute to the degradation of extracellular matrix during tumor invasion.

The phenomenon of acute phase (AP) response can be reproduced in vitro using cultured cells of hepatic origin by stimulation with the crude supernatant of activated monocytes (MoCM). Several monocyte-derived factors have been identified which might be responsible, alone or in combination, for the induction of AP response, but recently the attention has been focused on interleukin 6 (IL-6). We have previously shown that part of the AP response consists of the increase in the rate of transcription of the AP genes. Here we have treated the human hepatoma cell line Hep 3B with either crude MoCM or recombinant IL-6 and compared the effect of the two stimulants on the expression of both endogenous AP genes and recombinant plasmids introduced into the cells by transfection. The transfected plasmids contained the 5'-flanking region of AP genes fused to the coding region of the bacterial chloramphenicol acetyltransferase gene. We observe that the induction of mRNA accumulation of the endogenous genes corresponds to the transcriptional activation of the chloramphenicol acetyltransferase fusions. This is good evidence that the effect of IL-6 is totally or partially exerted at the level of transcription and that short segments of the 5'-flanking sequences of the inducible genes contain IL-6-responsive elements. Our results show that IL-6 is fully effective only on some of all the genes induced or repressed by MoCM, whereas others are only partially affected or totally nonresponsive.

The involvement of protein kinase C and its interaction with interleukin 1β in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short-term phorbol 12-myristate 13-acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12-myristate 13-acetate enhanced concentration- and time-dependently interleukin 6 release. This stimulatory action of phorbol 12-myristate 13-acetate was significantly reduced by staurosporine, by calphostin C, and by the desensitization of protein kinase C. Interleukin 1β increased interleukin 6 release in a concentration-related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1β stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1β (3 ng/ml) and phorbol 12-myristate 13-acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurosporine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C. Interleukin 1β stimulated interleukin 6 secretion via a mechanism that is also negatively modulated by a protein kinase C isoform or isoforms sensitive to staurosporine and desensitization. Finally, we showed that interleukin 1β and phorbol 12-myristate 13-acetate synergistically stimulated interleukin 6 release and its gene expression, operating in a manner insensitive to protein kinase C blockers and slightly reduced by protein kinase C desensitization.

Human C-Reactive protein (CRP) is inducible in liver cells during acute inflammation. Around 90 bp from the 5' flanking region of the human CRP gene contain, as shown here, information to induce the expression of a linked bacterial CAT gene specifically in human hepatoma (Hep3B) cells. The promoter is induced rapidly, faithfully and at high efficiency when transfected cells are exposed to conditioned medium from lipopolysaccharide stimulated peripheral monocytes. The sequences required for inducibility are located immediately upstream to the TATA element. A DNA segment from base -121 to -50 is capable of inducing transcription from the heterologous SV40 early promoter. Induction of CRP expression is probably exerted via the binding of at least one positive trans-acting factor.