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Human nucleolin (hNcl) is a multifunctional protein involved in the progression of various cancers and plays a key role in other pathologies. Therefore, there is still unsatisfied demand for hNcl modulators. Recently, we demonstrated that the plant ent-kaurane diterpene oridonin inhibits hNcl but, unfortunately, this compound is quite toxic for healthy cells. Trachylobane diterpene 6,19-dihydroxy-ent-trachiloban-17-oic acid (compound

According to the World Health Organization Cancer is the second leading cause of death globally. The methanol extracts of fourteen Middle-Eastern plants of the family Asteraceae were screened for antiproliferative activity on five cancer cell lines (A2780, MCF7, HeLa, RKO and Jurkat) by using MTT assay. Psiadia punctulata (DC.) Vatke was selected for isolation and elucidation of the bioactive constituents by 1D- and 2D-NMR, and MS analyses. Flow cytometry was used to evaluate cell cycle analysis, apoptotic hallmarks and reactive oxygen species. P. punctulata yielded a new sesquiterpene characterized as 7-hydroxy eudesman-3,5-dien-2-one (punctulin) (3) and three known sesquiterpenes: 1, 2 and 4. The antiproliferative activity of all sesquiterpenes was evaluated in Jurkat (T-cell leukemia) and HeLa cancer cell lines. Compound 1 (1β-hydroxy-8-oxo-cyperone) has induced a significant growth inhibition in Jurkat and HeLa cells (IC50 =12 and 18µM respectively). Flow cytometry of compound 1 has elucidated the mechanism of action by showing its ability to induce cell cycle arrest in Jurkat cells mainly in G0/G1 and, less markedly, in G2/M. Compound 1 also expressed strong antioxidant activity by reducing the basal level of peroxides DHFDA-load in Jurkat cells. Compound 1 antioxidant activity may have contributed to the observed cell cycle arrest.

The bioactive plant diterpene oridonin displays important pharmacological activities and is widely used in traditional Chinese medicine; however, its molecular mechanism of action is still incompletely described. In vitro and in vivo data have demonstrated anti-tumor activity of oridonin and its ability to interfere with several cell pathways; however, presently only the molecular chaperone HSP70 has been identified as a direct potential target of this compound. Here, using a combination of different proteomic approaches, innovative Cellular Thermal Shift Assay (CETSA) experiments, and classical biochemical methods, we demonstrate that oridonin interacts with Nucleolin, effectively modulating the activity of this multifunctional protein. The ability of oridonin to target Nucleolin and/or HSP70 could account for the bioactivity profile of this plant diterpene. Recently, Nucleolin has attracted attention as a druggable target, as its diverse functions are implicated in pathological processes such as cancer, inflammation, and viral infection. However, up to now, no small molecule as Nucleolin binders has been reported, thus our finding represents the first evidence of Nucleolin modulation by a small inhibitor.

The phytochemical investigation of Gymnocarpos decander aerial parts extract afforded two new saponins, 3-O-β-D-glucuronopyranosyl-2β,3β,16α,23-tetrahydroxyolean-12-en-28-O-β-D-apiofuranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl ester (1), 3-O-β-D-glucuronopyranosyl-2β,3β,16α-trihydroxyolean-12-en-28-O-α-L-rhamnopyranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl ester (2), and three new flavonol glycosides, isorhamnetin 3-O-2′′′′-O-acetyl−β-D-xylopyranosyl-(1 → 6)-[β-D-apiofuranosyl-(1 → 2)]-β-D-glucopyranoside (3), isorhamnetin 3-O-2‴-O-acetyl−β-D-xylopyranosyl-(1 → 6)-β-D-glucopyranoside (4), and quercetin 3-O-2‴-O-acetyl−β-D-xylopyranosyl-(1 → 6)-β-D-glucopyranoside (5), together with three known compounds. Their structures were determined by spectroscopic methods including 1D and 2D NMR analysis and high-resolution mass spectrometry. The new isolates were investigated for their potential cytotoxic activity on three cancer cell lines. Compounds 1 and 2 showed moderate antiproliferative activity.

Ten new (1-10) and six known (11-16) fusicoccane diterpenes were isolated from the roots of Hypoestes forsskaolii. The structural characterization of 1-10 was performed by spectroscopic analysis, including 1D and 2D NMR, ECD, and HRESIMS experiments. From a perspective of obtaining potential Hsp90α inhibitors, the isolates were screened by surface plasmon resonance measurements and their cytotoxic activity was assayed using Jurkat and HeLa cancer cells. Compound 6, 18-hydroxyhypoestenone, was shown to be the most active compound against Hsp90, and its interactions were studied also by biochemical and cellular assays and by molecular docking.

Brugada syndrome (BrS) is marked by coved ST-segment elevation and increased risk of sudden cardiac death. The genetics of this syndrome are elusive in over half of the cases. Variants in the SCN5A gene are the single most common known genetic unifier, accounting for about a third of cases. Research models, such as animal models and cell lines, are limited. In the present study, we report the novel NM_198056.2:c.1111C>T (p.Gln371*) heterozygous variant in the SCN5A gene, as well as its segregation with BrS in a large family. The results herein suggest a pathogenic effect of this variant. Functional studies are certainly warranted to characterize the molecular effects of this variant.

Eleven sesquiterpene lactones, including three new natural products (1-3), were isolated from the n-butanolic extract of Ambrosia cumanensis Kunth. aerial parts. The structure of all isolated compounds was elucidated by 1D- and 2D-NMR, and MS analyses. All compounds were tested for their antiproliferative activity on HeLa, Jurkat, and U937 cell lines. Compound 3, 2,3-dehydropsilostachyn C, showed cytotoxic activity with different potency in all cell lines. By means of flow cytometric studies, compound 3 was demonstrated to induce in Jurkat cells a G2/M cell cycle block, while in U937 elicited both cytostatic and cytotoxic responses.

Galactites is a genus of flowering plants belonging to Asteraceae family. This genus is mainly represented by the Galactites elegans (All.) Nyman ex Soldano, the milky thistle, a plant of Mediterranean origin. Galactites elegans is consumed as a monofloral boar thistle honey. Chromatography separation of CHCl3 and n-BuOH extracts of aerial parts of G. elegans led to isolation of 18 pure compounds. Their structures were elucidated by 1D-and 2D-NMR spectroscopy and confirmed by mass spectrometry analysis. Sinapic aldehyde, abietin, chlorogenic acid, neochlorogenic acid, 8α-hydroxypinoresinol, 9α-hydroxypinoresinol, pinoresinol, 4-ketopinoresinol, nortrachelogenin, and erythro-guaiacylglycerol-β-O-4′-dihydroconiferyl alcohol were isolated from CHCl3 extract, while luteolin 4′-O-glucuronide, naringenin-7-O-neohesperidoside, kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside, apigenin-7-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside, quercitrin, quercetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside, ciwujiatone, and nortrachelogenin-4,4′-di-O-β-D-glucopyranoside were obtained from n-BuOH extract. The majority of isolated compounds displayed a significant antioxidant potential in vitro test (DPPH). The ability of compounds to reduce the level of peroxides in control and BHP-treated Jurkat cells was studied. The lignan derivatives were also able to reduce at 50 μM the basal level of peroxides in Jurkat cells as well as counteract peroxide increase induced by BHP treatment. Particularly 8α-hydroxypinoresinol was the most active showing 70% of peroxide level inhibition.

The aerial parts of Guarea guidonia afforded three new tirucallane-type triterpenoids: 3,4-seco-tirucalla-4(28),8(9),24(25)-trien-7α,11α-dihydroxy-21,23-epoxy-3,11-olide, named guareolide (1), 3,4-seco-tirucalla-4(28),7(8),24(25)-trien-21-hydroxy-21,23-epoxy-3-oic acid, named guareoic acid A (2), and 3,4-seco-tirucalla-4(28),7(8),24(25)-trien-21,23-epoxy-3-oic acid, named guareoic acid B (3), of which 1 possessed an unusual seven-membered lactone ring. Seven known terpenes were also isolated and characterized as flindissone, 7-acetyldihydronomilin, picroquassin E, boscartol C, and cneorubins A, B, and X. Their structures were determined by spectroscopic methods including one-dimensional and two-dimensional nuclear magnetic resonance analysis and high-resolution mass spectrometry. The isolates were investigated for their potential cytotoxic activity on Jurkat, HeLa, and MCF7 cancer cell lines. Flindissone and compound 2 showed an antiproliferative activity in all cell lines. Further studies revealed that flindissone, the most active compound, induced in Jurkat and HeLa cells both cytostatic and cytotoxic responses.

Background. Oxidative stress is a recognized pathogenic mechanism in chronic obstructive pulmonary disease (COPD). Expression of the NAD+-dependent deacetylase Sirtuin 1 (SIRT1), an antiaging molecule with a key role in oxidative stress response, has been described as decreased in the lung of COPD patients. No studies so far investigated whether systemic SIRT1 activity was associated to decreased lung function in this disease. Methods. We measured SIRT1 protein expression and activity in peripheral blood mononuclear cells (PBMCs) and total oxidative status (TOS), total antioxidant capacity (TEAC), and oxidative stress index (TOS/TEAC) in the plasma of 25 COPD patients, 20 healthy nonsmokers (HnS), and 20 healthy smokers (HS). Results. The activity of SIRT1 was significantly lower in COPD patients compared to both control groups while protein expression decreased progressively (HnS > HS > COPD). TOS levels were significantly lower in HnS than in smoke-associated subjects (COPD and HS), while TEAC levels were progressively lower according (HnS > HS > COPD). In COPD patients, SIRT1 activity, but not protein levels, correlated significantly with both lung function parameters (FEV1/FVC and FEV1) and TEAC. Conclusions. These findings suggest loss of SIRT1-driven antioxidant activity as relevant in COPD pathogenesis and identify SIRT1 activity as a potential convenient biomarker for identification of mild/moderate, stable COPD.

A pentacyclic triterpene, named salvibuchanic acid (1), together with five known compounds, were isolated from the roots of Salvia buchananii Hedge (Lamiaceae). The structural characterisation of all compounds was performed by spectroscopic analyses, including 1D and 2D NMR and HRESIMS experiments. The lupane triterpene (1) and hyptadienic acid (2) were investigated for their potential cytotoxic activity on Jurkat, HeLa and MCF7 cell lines. Both compounds showed an interesting antiproliferative activity with similar potency in all cell lines. By means of flow cytometric studies, hyptadienic acid (2) induced in HeLa cells a S cell cycle block, while 1 elicited both cytostatic and cytotoxic responses.

Objectives: The aim of this study was to investigate anti-leukaemic potential of coronopilin, a sesquiterpene lactone from Ambrosia arborescens, and to characterize mechanism(s) underlying its activity. Materials and methods: The study was conducted on Jurkat and U937, two leukaemia-derived cell lines. Apoptosis and impairment of cell cycle progression were evaluated by flow cytometry and by microscopic analysis. Changes in protein expression and activation were evaluated by western blot analysis. Coronopilin-tubulin covalent adducts were demonstrated by mass spectrometry. Results: Coronopilin inhibited (IC50 ≤ 20 μm) leukaemia cell population growth, but displayed poor cytotoxicity to normal white blood cells. On Jurkat cells, coronopilin exerted cell population growth inhibition activity, mainly by triggering caspase-dependent apoptosis. Conversely, in U937 cells, coronopilin’s primary response was a robust arrest in G2/M. Marked increase in mitotic index and presence of activated cyclin B1/Cdk1 complex, phosphorylated histone H3 at Ser10, and hyperpolymerized tubulin indicated that cells accumulated in mitosis. Prolonged mitotic arrest ultimately resulted in U937 mitotic catastrophe, and dying cells exhibited the features of non-caspase-dependent death. Conclusions: This study demonstrated that coronopilin efficiently inhibited leukaemia cell population growth by triggering cell type-specific responses. Moreover, coronopilin-mediated cell population expansion inhibition was specific to neoplastic cells, as normal white blood cell viability was not significantly affected. Thus, coronopilin may represent an interesting new chemical scaffold upon which to develop new anti-leukaemic agents.

One new phenolic compound (1) together with nine known derivatives were isolated from the aerial part apolar extracts of Calligonum azel Maire and Calligonum comosum L'Hér (Polygonaceae). Their structures were established on the basis of 1D and 2D NMR spectroscopy, as well as ESI-MS analysis. The anti-inflammatory and antioxidant potential of pure compounds was evaluated in J774A.1 murine macrophages and Jurkat cells. Among tested molecules, 4-ethoxy-1,2-benzendiol, tamgermanetin, and α,β-diamino-4-hydroxybenzene butanoic acid exerted the more interesting activity.

The growth temperature adaptation of six model proteins has been studied in 42 microorganisms belonging to eubacterial and archaeal kingdoms, covering optimum growth temperatures from 7 to 103 degrees C. The selected proteins include three elongation factors involved in translation, the enzymes glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase, the cell division protein FtsZ. The common strategy of protein adaptation from cold to hot environments implies the occurrence of small changes in the amino acid composition, without altering the overall structure of the macromolecule. These continuous adjustments were investigated through parameters related to the amino acid composition of each protein. The average value per residue of mass, volume and accessible surface area allowed an evaluation of the usage of bulky residues, whereas the average hydrophobicity reflected that of hydrophobic residues. The specific proportion of bulky and hydrophobic residues in each protein almost linearly increased with the temperature of the host microorganism. This finding agrees with the structural and functional properties exhibited by proteins in differently adapted sources, thus explaining the great compactness or the high flexibility exhibited by (hyper)thermophilic or psychrophilic proteins, respectively. Indeed, heat-adapted proteins incline toward the usage of heavier-size and more hydrophobic residues with respect to mesophiles, whereas the cold-adapted macromolecules show the opposite behavior with a certain preference for smaller-size and less hydrophobic residues. An investigation on the different increase of bulky residues along with the growth temperature observed in the six model proteins suggests the relevance of the possible different role and/or structure organization played by protein domains. The significance of the linear correlations between growth temperature and parameters related to the amino acid composition improved when the analysis was collectively carried out on all model proteins.

In human cancer cells, BAG3 protein is known to sustain cell survival. Here, for the first time, we demonstrate the expression of BAG3 protein both in equine sarcoids in vivo and in EqS04b cells, a sarcoid-derived fully transformed cell line harbouring bovine papilloma virus (BPV)-1 genome. Evidence of a possible involvement of BAG3 in equine sarcoid carcinogenesis was obtained by immunohistochemistry analysis of tumour samples. We found that most tumour samples stained positive for BAG3, even though to a different grade, while normal dermal fibroblasts from healthy horses displayed very weak staining pattern for BAG3 expression. By siRNA technology, we demonstrate in EqS04b the role of BAG3 in counteracting basal as well as chemical-triggered pro-death signals. BAG3 down-modulation was indeed shown to promote cell death and cell cycle arrest in G0/G1. In addition, we found that BAG3 silencing sensitized EqS04b cells to phenethylisothiocyanate (PEITC), a promising cancer chemopreventive/chemotherapeutic agent present in edible cruciferous vegetables. Notably, such a pro-survival role of BAG3 was less marked in E. Derm cells, an equine BPV-negative fibroblast cell line taken as a normal counterpart. Altogether our findings might suggest a mutual cooperation between BAG3 and viral oncoproteins to sustain cell survival.

Eight new limonoids (1–8) and one new phenol glycoside (9), along with six known compounds, were isolated from the leaves of Azaridachta indica. The structures of 1–9 were elucidated on the basis of spectroscopic data analysis. Compounds isolated were assayed for their cytotoxicity against different cancer cell lines. Moreover, their ability to interact with the molecular chaperone Hsp90, affecting its biological activity, was tested.

Highlights • BAG3 down-modulation by siRNA technology restored p53. • Reduced BAG3 expression sensitized HeLa but not C33A (HPV-negative) cells to PEITC. • Reduced BAG3 expression was associated with a decrease of E6 viral protein levels., BAG3 is a multi-functional component of tumor cell pro-survival machinery, and its biological functions have been largely associated to proteasome system. Here, we show that BAG3 down-modulation resulted in reduced cell viability and enhanced PEITC-induced apoptosis largely more extensively in HeLa (HPV18+) rather than in C33A (HPV−) cervical carcinoma cell lines. Moreover, we demonstrate that BAG3 suppression led to a decrease of viral E6 oncoprotein and a concomitant recovery of p53 tumor suppressor, the best recognized target of E6 for proteasome degradation. E6 and p53 expression were modulated at protein level, since their respective mRNAs were unaffected. Taken together our findings reveal a novel role for BAG3 as host protein contributing to HPV18 E6-activated pro-survival strategies, and suggest a possible relevance of its expression levels in drug/radiotherapy-resistance of HPV18-bearing cervical carcinomas.

The semi-synthetic ent-kaurane 15-ketoatractyligenin methyl ester (SC2017) has been previously reported to possess high antiproliferative activity against several solid tumor-derived cell lines. Our study was aimed at investigating SC2017 tumor growth-inhibiting activity and the underlying mechanisms in Jurkat cells (T-cell leukemia) and xenograft tumor models. METHODS: Cell viability was evaluated by MTT assay. Cell cycle progression, reactive oxygen species (ROS) elevation and apoptotic hallmarks were monitored by flow cytometry. Inhibition of thioredoxin reductase (TrxR) by biochemical assays. Levels and/or activation status of signaling proteins were assessed by western blotting. Xenograft tumors were generated with HCT 116 colon carcinoma cells. RESULTS: SC2017 displayed cell growth-inhibiting activity against Jurkat cells (half maximal inhibitory concentration values (IC50)

Oridonin, an ent-kaurane diterpene isolated from well known Chinese medicinal plant Isodon rubescens, has been shown to have multiple biological activities. Among them, the anticancer activity has been repeatedly reported by many research groups. The chemopreventive and antitumor effects of oridonin have been related to its ability to interfere with several pathways which are involved in cell proliferation, cell cycle arrest, apoptosis and/or autophagy. Despite the number of studies performed on this diterpene, the molecular mechanism underlying its cellular activity remains to be elucidated. Hence, we tried to mine target protein(s) of oridonin by employing a mass spectrometry-based chemical proteomics approach, providing evidences that oridonin is able to directly bind the multifunctional, stress-inducible heat shock protein 70 1A (HSP70 1A). Oridonin/HSP70 complex formation was confirmed in leukemia-derived Jurkat cells. The characterization of HSP70 inhibition by oridonin was performed using chemical and biological approaches. Moreover, the binding site of oridonin on the chaperone was identified by a mass-based approach combined with Molecular Dynamics simulations. Biological significance Although natural products showed high efficiency and several of these agents have now entered in clinical trials, information concerning the mechanisms of action at a molecular level of many of them is very poor or completely missed. Nevertheless, the identification of the molecular target of a drug candidate has several advantages. The most significant is the ability to set up target-based assays and to allow structure–activity relationship studies to guide medicinal chemistry efforts towards lead optimization. The knowledge of drug targets can also facilitate the identification of potential toxicities or side effects, if there is any precedent of toxicities for the identified target. Achieving this in an effective, unbiased and efficient manner subsists as a significant challenge for the new era in drug discovery and optimization. In the present study, we used a chemical proteomic approach aimed to define the possible protein target of the ent-kaurane diterpene oridonin. This natural compound has drawn a rising attention for cancer biologists due to its remarkable anti-tumor activities: accumulating evidence has suggested that oridonin is able to hamper the progression of tumor, mitigate tumor burden and alleviate cancer syndrome, which may improve greatly the survival rates of cancer patients; however molecular mechanisms by which this compound exerts its anti-tumor activities still remained to be discovered. We identified the molecular chaperone HSP70 1A as an oridonin target in Jurkat cells, thus suggesting a mechanism of action for the diterpene consistent with the multiple biological activities described for it. HSP70 inhibition by oridonin might indeed simultaneously result in the impairment of some of client proteins, thus in turn affecting several molecular pathways. Shedding light on the molecular basis of the biological activity of oridonin, our findings may be relevant for possible therapeutic applications of oridonin, such as its use in combination and the design of new therapeutic approaches. In addition, this research demonstrates the effectiveness of chemical proteomic approaches in drug discovery studies and in orphan drug molecular target identification.

The potential of heat shock protein 90 (Hsp90) as a therapeutic target for numerous diseases has made the identification and optimization of novel Hsp90 inhibitors an emerging therapeutic strategy. A surface plasmon resonance (SPR) approach was adopted to screen some iridoids for their Hsp90 α binding capability. Twenty-four iridoid derivatives, including 13 new natural compounds, were isolated from the leaves of Tabebuia argentea and petioles of Catalpa bignonioides. Their structures were elucidated by NMR, electrospray ionization mass spectrometry, and chemical methods. By means of a panel of chemical and biological approaches, four iridoids were demonstrated to bind Hsp90 α. In particular, the dimeric iridoid argenteoside A was shown to efficiently inhibit the chaperone in biochemical and cellular assays. Our results disclose C9-type iridoids as a novel class of Hsp90 inhibitors.

Four new clerodane diterpenes (1–4) and one new phenylpropanoid (5) have been isolated from Clerodendrum splendens aerial parts, together with nine known compounds. Their structures were determined by spectroscopic and spectrometric analysis and chemical methods. The absolute configuration of the 15,16-diol moiety in 4 was determined by Snatzkeʼs method. Antiproliferative activity of diterpenes in HeLa cells was also evaluated. The IC50 values were 98 ± 11 µM for 3 and 101 ± 8 µM for 1, respectively.

The endogenous components of the thioredoxin system in the Antarctic eubacterium Pseudoalteromonas haloplanktis have been purified and characterised. The temperature dependence of the activities sustained by thioredoxin (PhTrx) and thioredoxin reductase (PhTrxR) pointed to their adaptation in the cold growth environment. PhTrxR was purified as a flavoenzyme and its activity was significantly enhanced in the presence of molar concentration of monovalent cations. The energetics of the partial reactions leading to the whole electron transfer from NADPH to the target protein substrate in the reconstituted thioredoxin system was also investigated. While the initial electron transfer from NADPH to PhTrxR was energetically favoured, the final passage to the heterologous protein substrate enhanced the energetic barrier of the whole process. The energy of activation of the heat inactivation process essentially reflected the psychrophilic origin of PhTrxR. Vice versa, PhTrx possessed an exceptional heat resistance (half-life, 4.4 h at 95 °C), ranking this protein among the most thermostable enzymes reported so far in psychrophiles. PhTrxR was covalently modified by glutathione, mainly by its oxidised or nitrosylated forms. A mutagenic analysis realised on three non catalytic cysteines of the flavoenzyme allowed the identification of C(303) as the target for the S-glutathionylation reaction.

Angiogenesis is a crucial step in many pathological conditions like cancer, inflammation and metastasis formation; on these basis the search for antiangiogenic agents has widened. In order to identify new compounds able to interfere in the Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1, also known as Flt-1) recognition by VEGFs family members, we screened Calycolpus moritzianus (O. Berg) Burret leaves extracts by a competitive ELISA-based assay. MeOH and CHCl3 extracts and several their fractions demonstrated to be able to prevent VEGF or PlGF interaction with Flt-1, with an inhibition about 50% at concentration of 100 μg/mL. Phytochemical and pharmacological investigation of the active fractions led to the isolation of flavonoids, and terpenes.

Rimonabant (SR141716), a cannabinoid CB1 receptor antagonist known for anti-obesity activity, has more recently been shown to inhibit tumor cell growth. Here we demonstrated the antitumor potential of SR141716 in leukemia-derived cell lines and its low toxicity in normal cells (PBMC). SR141716 (1-20 mu M range of doses) reduced Jurkat and U937 cell number by activating death signals as well as affecting cell cycle progression. The most prominent response in U937 to SR141716 was a G(0)/G(1) block, while in Jurkat cells there was activation of cell death processes. SR141716-treated cells exhibited the morphological and biochemical features of apoptosis and to some extent necrosis. Apoptotic mode of cell death was confirmed in both cell lines by analysis of cell morphology, phosphatidylserine exposure and DNA fragmentation. Moreover, the drug was found to induce an early and robust mitochondrial membrane depolarization. In Jurkat cells the apoptotic process was typically caspase-dependent, while in U937 caspase-independent pathways were also activated. The contribution of PARP activation to SR141716-induced apoptosis in U937 was suggested by protein PARylation, AIF release and apoptosis reversal by PARP inhibitors. Moreover. SR141716 negatively modulated, especially in U937, the PI3K/AKT pathways. In conclusion, our data indicate that SR141716 elicits alternative response and/or cell death pathways depending on the cell type affected. (C) 2010 Elsevier Inc. All rights reserved.

Thioredoxin and thioredoxin reductase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis were obtained as recombinant His-tagged proteins (rPhTrx and rPhTrxR, respectively). rPhTrxR is organised as a homodimeric flavoenzyme, whereas rPhTrx is a small monomeric protein, both containing a functional disulfide bridge. However, three additional cysteines are present as free thiols in purified rPhTrxR. When individually tested in specific assays, rPhTrxR and rPhTrx display a full activity at low temperatures, an indispensable requirement for cold-adapted proteins. In particular, rPhTrxR catalyses the NADPH dependent reduction of DTNB and rPhTrx provokes the insulin precipitation in the presence of DTT. The analysis of the effect of temperature on these reactions indicates that rPhTrxR is more cold-adapted than rPhTrx, having a higher psychrophilicity. The combined activity of rPhTrxR and rPhTrx, tested in a reconstituted assay containing NADPH as electrons donor and human insulin as the thioredoxin substrate, demonstrates a direct functional interaction between the purified recombinant components of the thioredoxin system of P. haloplanktis. Furthermore, the NADPH-dependent reduction of rPhTrx catalysed by rPhTrxR is fully reversible and allows the determination of its redox potential, whose value is in the range of other bacterial and archaeal thioredoxins. The analysis of the thermostability of rPhTrxR points to its discrete heat resistance. However, rPhTrx is much more heat resistant, with a half inactivation time of about 4 h at 95 °C. This exceptional heat resistance for a psychrophilic protein is significantly decreased by the reduction of the disulfide bridge of rPhTrx. Functionality, thermodependence and thermostability of the P. haloplanktis thioredoxin system point to the relevance of this key mechanism for the preservation of the reduced state of cytoplasmic proteins even in a cold-adapted source. © The Royal Society of Chemistry 2009.

The role of three amino acid residues (Q143, Y34, S82) of rat mitochondrial superoxide dismutase (ratSOD2) in the enzymatic activity, thermostability, and post-translational modification of the enzyme was investigated through site-directed mutagenesis studies. Six recombinant forms of the enzyme were produced, carrying the Q143 or H143 residue with or without the Y34F or S82A replacement. All proteins bound manganese as active cofactor and were organized as homotetramers. The greatest effect on the activity (sixfold reduction) was observed in ratSOD2 forms containing the H143 variant, whereas Y34F and S82A substitutions moderately reduced the enzymatic activity compared to the Q143 form. Heat inactivation studies showed the high thermo-tolerance of ratSOD2 and allowed an evaluation of the related activation parameters of the heat inactivation process. Compared to Q143, the H143 variant was significantly less heat stable and displayed moderately lower enthalpic and entropic factors; the Y34F substitution caused a moderate reduction of heat stability, whereas the S82A replacement slightly improved the thermo-tolerance of the Q143 variant; both substitutions significantly increased enthalpic and entropic factors of heat inactivation, the greatest effect being observed with S82A substitution. All recombinant forms of ratSOD2 were glutathionylated in Escherichia coli, a feature pointing to the high reactivity of ratSOD2 toward glutathione. Moreover, the S82 position of the enzyme was phosphorylated in an in vitro system containing human mitochondrial protein extracts as source of protein kinases. These data highlight the role played by some residues in ratSOD2 and suggest a fine regulation of the enzyme occurring in vivo. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 1215–1226, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

A new oleanolic acid triterpene glucoside, 3- O-β-D-glucopyranosyl-3β,21β,28-trihydroxy-olean-12-en-27-oic acid (1), has been isolated together with twelve known compounds from the chloroform and ethyl acetate extracts of Genista numidica Spach (Fabaceae) aerial parts. The structures were elucidated by spectroscopic and spectrometric analyses, mainly 1D-, 2D-NMR and MS data, and comparison with the literature. The antiproliferative activity of isolates was investigated on Jurkat, HeLa, and MCF7 cell lines. The most active triterpene, 3- O-β-D-glucopyranosyl-olean-12-en-3β,27,28,29-tetraol, showed activity in all cell lines. Further studies revealed that this compound induced in HeLa cells a cytostatic response.