Rigini M. Papi

Diorganotin(IV) complexes of dipeptides containing the α-aminoisobutyryl residue (Aib): Preparation, structural characterization, antibacterial and antiproliferative activities of [(n-Bu)2Sn(H−1L)] (LH = H-Aib-L-Leu-OH, H-Aib-L-Ala-OH)

Two new organotin(IV) complexes with dianionic dipeptides containing the alpha-aminoisobutyryl residue (Aib) as ligands are described. The solid complexes [(n-Bu)(2)Sn(H(-1)L(A))] x 2MeOH (1 x 2MeOH) (L(A)H=H-Aib-L-Leu-OH) and [(n-Bu)(2)Sn(H(-1)L(B))] x MeOH (2 x MeOH) (L(B)H=H-Aib-L-Ala-OH) have been isolated and characterized by single-crystal X-ray crystallography and spectroscopic techniques (H(-1)L(2-) is the dianionic form of the corresponding dipeptide). Complexes 1 x 2MeOH and 2 x MeOH are monomeric with similar molecular structures. The doubly deprotonated dipeptide behaves as a N(amino), N(peptide), O(carboxylate) ligand and binds to the Sn(IV) atom. The five-coordinate metal ion has a distorted trigonal bipyramidal geometry. A different network of intermolecular hydrogen bonds in each compound results in very dissimilar supramolecular features. The IR, far-IR, Raman and (119)Sn NMR data are discussed in terms of the nature of bonding and known structures. The antibacterial and antiproliferative activities as well as the effect of the new compounds on pDNA were examined. Complexes 1 and 2 are active against the gram-positive bacteria Bacillus subtilis and Bacillus cereus. The IC(50) values reveal that the two compounds express promising cytotoxic activity in vitro against a series of cell lines.

Encapsulated Escherichia coli in alginate beads capable of secreting a heterologous pectin lyase

BackgroundProduction of heterologous proteins in the E. coli periplasm, or into the extracellular fluid has many advantages; therefore naturally occurring signal peptides are selected for proteins translocation. The aim of this study was the production in high yields of a recombinant pectin lyase that is efficiently secreted and the encapsulation of transformed E. coli cells for pectin degradation in a biotechnological process.ResultsThe nucleotide sequence of Bacillus subtilis α-amylases signal peptide was fused to the N-terminal of an heterologously expressed pectin lyase in E. coli BL21 [DE3]. Thus pectin lyase secretion was achieved into the extracellular growth medium. E. coli cells harboring the recombinant plasmid heterologously express pectin lyase to around 22% of the total cellular proteins, as it was estimated by SDS-PAGE and image analysis. IPTG induces the heterologously expressed enzyme, which is initially distributed extracellularly (7 hour) and later on at the periplasmic (9 hours) or cytosolic fraction (20 hours). No pectin lyase activity was found in the membranes fraction and in the inclusion bodies. Encapsulation of the recombinant strains of E. coli in alginate or alginate/silica beads 1:5 showed that pectin lyase could degrade effectively its substrate, for at least ten operational cycles.ConclusionSecretion of an heterologously overexpressed pectin lyase in E. coli BL21 [DE3] was achieved in this study. For this purpose the signal peptide of α-amylase from B. subtilis was fused to the N-terminal domain of pectin lyase. Encapsulated E. coli BL21 [DE3] cells harboring pET29c/exPNL were used successfully for pectin degradation up to ten operational cycles indicating that under special conditions this might have biotechnological implementations.

Mannose binding lectin and ficolin-2 polymorphisms are associated with increased risk for bacterial infections in children with B acute lymphoblastic leukemia.

We aimed to investigate whether the presence of mannose binding lectin (MBL2), ficolin 2 (FCN2) polymorphisms or the combined deficiency significantly influence the risk and subsequently the frequency of chemotherapy‐induced bacterial infections in children with B acute lymphoblastic leukemia (B‐ALL).

The synthesis of 1,3,5‐triazine derivatives and JNJ7777120 analogues with histamine H4 receptor affinity and their interaction with PTEN promoter

The involvement of histamine and H4 receptor (H4R) in cancer has been investigated recently using the H4R agonists and antagonists. The scope of the research project was synthesis and exploration of the consequences of a group of compounds with histamine H4 receptor (H4R) affinity on the promoter of PTEN gene encoding the antitumor PTEN protein. The series of novel compounds based either on H4R antagonists JNJ7777120 structure or 1,3,5‐triazine scaffold were synthesized, evaluated for histamine H4R affinity and used in this study. Compounds 5 and 7 belonging to the group of JNJ7777120 analogues showed the highest interaction with the promoter of PTEN gene and weak affinity against H4R with Ki value >100 μm. These compounds showed no significant effect on neuroblastoma IMR‐32 cells viability indicating no correlation between PTEN gene promoter affinity and antitumor activity. Compound 6, another JNJ7777120 analogue, showed the highest effect on IMR‐32 viability with calculated IC50 = 23.27 μm. The 1,3,5‐triazine derivatives exhibited generally low or medium interaction with PTEN gene promoter. However, the 1,3,5‐triazine derivative 11 with the para‐bromo substituent showed the highest affinity against H4R with Ki value of 520 nm and may be considered as a new lead structure.

Overexpression of the pectin lyase gene of Pseudomonas marginalis in Escherichia coli and purification of the active enzyme.

A pectin lyase gene (pnl) of Pseudomonas marginalis was cloned and overexpressed in Escherichia coli BL21(DE3). The pnl gene was amplified by PCR, inserted into pET29c with a six‐His tag and the overproduced active enzyme was purified almost to homogeneity using a Ni2+‐nitrilotriacetate–agarose column. The purified pectin lyase (PNL; EC 4.2.2.10, family 1) is inhibited by NAD+ (at concentrations above 0.25 mM), NADH or dithiothreitol. Evidence for the existence of a heat‐labile protein inhibitor of PNL is also reported. The DNA‐binding ability of PNL was demonstrated by DNA‐retardation experiments. The partially purified enzyme was incubated with plasmid DNA and the complex was shifted to a higher molecular mass. Analysis of the electroeluted proteins from the protein–DNA complex revealed that one of the electroeluted protein bands was PNL. Antibodies against the overexpressed PNL were also prepared and partially purified.

Lipase activity in Thermus thermophilus HB8: Purification and characterization of the extracellular enzyme

In this study, the lipolytic activity of Thermus thermophilus HB8 was examined. The addition of various oils increased the production of extracellular lipolytic activity, while a combination of olive oil and glucose increased both extracellular and intracellular lipolytic activity. The oxygen transfer rate had a significant influence on both biomass and production of extra- or intra-cellular lipolytic activity. The formation of white halos due to the hydrolysis of oleic acid ester (Tween 80) in agar plates containing Nile Blue and the formation of Ca2+-oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular extract were electrophoresed under denatured and renatured conditions, using α-naphthyl acetate and Fast Blue RR, major bands at 56 kDa or 62 and 32 kDa were observed, respectively. The 56 kDa extracellular enzyme was partial purified and characterized. Its peak of activity occurred at 80°C and pH 7.0, while the T1/2 was 1 h at 100°C. The Km of the partial purified enzyme was 1 mM and the Vmax was 0.044 U/mL/min when using p-nitrophenyl laurate as substrate. The presence of Ca2+ and Hg2+ stimulated lipase activity, whereas Zn2+, Co2+, or EDTA inhibited lipase activity. The highest activity was observed in the presence of coconut oil and p-nitrophenyl laurate (pNPL). Purified lipase was the most stable in the presence of various organic solvents, such as pentanol, chloroform and n-dodecane. Because of the superior thermostability and stability in the presence of organic solvents of T. thermophilus extracellular lipase, this lipase holds great promise for use in industrial applications.

Synthesis, Crystal Structures, and DNA Binding Properties of Zinc(II) Complexes with 3-Pyridine Aldoxime

The employment of 3-pyridine aldoxime, (3-py)CHNOH, in ZnII chemistry has afforded two novel compounds: [Zn(acac)2{(3-py)CHNOH}]·H2O (1·H2O) [where acac− is the pentane-2,4-dionato(-1) ion] and [Zn2(O2CMe)4{(3-py)CHNOH}2] (2). Complex 1·H2O crystallizes in the monoclinic space group P21/n. The ZnII ion is five-coordinated, surrounded by four oxygen atoms of two acac− moieties and by the pyridyl nitrogen atom of the (3-py)CHNOH ligand. Molecules of 1 interact with the water lattice molecules forming a 2D hydrogen-bonding network. Complex 2 crystallizes in the triclinic P-1 space group and displays a dinuclear paddle-wheel structure. Each ZnII exhibits a perfect square pyramidal geometry, with four carboxylate oxygen atoms at the basal plane and the pyridyl nitrogen of one monodentate (3-py)CHNOH ligand at the apex. DNA mobility shift assays were performed for the determination of the in vitro effect of both complexes on the integrity and the electrophoretic mobility of pDNA.

Dinuclear copper(I) complexes of N-methylbenzothiazole-2-thione: synthesis, structures, antibacterial activity and DNA interaction

Abstract Three copper(I) halide complexes containing N-methylbenzothiazole-2-thione (mbtt) and triphenylphosphine (PPh3) have been prepared and structurally characterized by X-ray single-crystal analysis. Copper(I) halide precursors [CuΧ(PPh3)]4 (X = Cl, Br, I) react with mbtt in 1 : 4 M ratio to give complexes of formula [CuΧ(mbtt)(PPh3)]2. Hereby, dimerization is achieved in case of copper(I) chloride and bromide via halide bridges, while copper(I) iodide gives the binuclear thione-S-bridged dimer. The new complexes show moderate in vitro antibacterial activity against certain bacterial strains. The interaction of the compounds with calf-thymus DNA was monitored via UV–vis spectroscopy, DNA-viscosity measurements and their competition with ethidium bromide for the DNA intercalation sites studied by fluorescence emission spectroscopy. Intercalation was revealed as the probable mode of binding.

Genetic variation of Fraxinus angustifolia natural populations in Greece based on nuclear and chloroplast microsatellite markers

Assessment of genetic variation within and among populations is an essential parameter for the effective conservation of forest genetic resources. In this work, the genetic diversity within and among natural populations of the forest tree species Fraxinus angustifolia Vahl in Greece was studied using selected nuclear and chloroplast microsatellite DNA loci. Eight natural populations of F. angustifolia were identified in different locations of the mainland of Greece, and a total of 230 individuals were studied. High polymorphism was observed within populations, while the genetic differentiation among populations was moderate. Intra-population diversity was correlated with geographical coordinates, but no isolation by distance was observed. Of the three haplotypes identified, only one was dominant. Putative ancestral haplotypes were found at small spatial scales suggesting that population expansions could have originated in the region. This study located in sympatry haplotypes that in other parts of Europe are in allopatry, reinforcing the notion of population expansions from the south of Balkans including Greece. Suggestions for conservation and management of F. angustifolia are also reported.

Identification of PHA loci in Thermus thermophilus HB8 genome

Withdrawn PP2B-18 Co-ordinate induction of PPARa and SREBP2 in multifunctional protein 2 deficient mice K. Martens, E. Ver Loren Van Themaat, P. Van Veldhoven, A. Van Kampen and M. Baes Laboratory for Cell Metabolism and LIPIT, K. U. Leuven, BELGIUM, Bioinformatics Laboratory, AMC, Amsterdam, NETHERLANDS Introduction: Mice with inactivation of the D-specific multifunctional protein 2 (MFP2), a crucial enzyme of peroxisomal b-oxidation, develop multiple pathologies in diverse tissues already starting in the post-natal period and causing death before the age of 6 months. Methods and results: Gene expression profiling performed on liver of 2-day-old pups revealed up-regulation of PPARa responsive genes in knockout mice. Surprisingly, also genes involved in cholesterol biosynthesis were markedly induced. Real-time PCR confirmed the induction of PPARa target genes and of HMGCR and SREBP2, both involved in cholesterol synthesis, as well in 2-day-old pups as in adults. In accordance, the rate of cholesterol biosynthesis was markedly increased in liver of knockout mice but the hepatic cholesterol concentration was unaltered. In PPARa/MFP2 double knockout mice, upregulations of SREBP2 and HMGCR were less pronounced suggesting a link between PPARa induction and cholesterol synthesis. Conclusion: These data indicate that impaired peroxisomal b-oxidation causes an accumulation of PPARa ligands in the intact animal and a concomitant up-regulation of genes of cholesterol biosynthesis through increased expression of SREBP2. Since the hepatic cholesterol concentration was not different between the genotypes, it appears that the up-regulation was not triggered by a reduced level of cholesterol, neither resulted in increased cholesterol levels. PP2B-19 Dominant negative mutant of CREB inhibits TrkCinduced activation of the nur77 promoter J. Matuszyk and D. Klopotowska L.Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, POLAND nur77 (also termed NGFI-B, TR3, NR4A1) and its family members Nurr1 and Nor-1 are orphan nuclear receptors that play important roles in neuronal differentiation, memory consolidation, stress response, and apoptosis. The promoter region of the nur77 gene contains four near AP-1 (NAP) sites 5’-TGCGTCA. Results of Yoon & Lau [1] supported the hypothesis that JunD, but not CREB, binding to two proximal NAP elements is responsible for induction of the transcription of nur77 in response to nerve growth factor. However, the results of the present study indicate that A-CREB (the dominant negative mutant of CREB) inhibits the activation of the nur77 promoter in response to the activation of TrkC. In conclusion it is suggested that activation of the nur77 promoter in response to neurotrophins requires the co-operation of both CREB and JunD. Reference: 1. Yoon & Lau, MCB 1994; 14: 7731–7743. PP2B-20 Genome-wide location of glucocorticoid receptor binding sites D. Mitsiou, M. McCalman, M. Alexis and H. Stunnenberg Molecular Endocrinology Programme, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, GREECE, NCMLS, Department of Molecular Biology, Radboud University of Nijmegen, Nijmegen, NETHERLANDS Introduction: Glucocorticoids (GCs) are essential steroid hormones that regulate a variety of physiological processes including growth, differentiation, programmed cell death, glucose homeostasis and protein, lipid and carbohydrate metabolism. GCs exert their functions through the glucocorticoid receptor (GR), a ligand inducible transcription factor that binds to a variety of promoter elements, including glucocorticoid response elements (GREs), and regulates gene transcription in a cell-specific manner. The mechanisms by which GR selectively regulates cell-specific transcription and the global networks regulated by GR are not well established. In the present study we mapped the human GR binding sites on a genome-wide scale. Methods: To identify the global profile of GR binding sites we used chromatin immunoprecipitation (ChIP), with a monoclonal antibody against GR, combined with either hybridization on genomic microarrays covering the entire genome (ChIP-on-chip) or massively parallel sequencing (ChIP-seq). Results: We mapped the genome-wide GR binding sites in immortalized human hepatocytes (IHH) and HeLa cells and identified known and novel cis regulatory elements and target genes in cell-specific contexts. This study revealed the presence of GR binding sites in previously un-explored regions of the genome as well as networks of transcription factors underlying glucocorticoid signalling. Our data demonstrated distinct mechanisms of glucocorticoid-mediated gene regulation and shed light on the global networks modulated by GR. Conclusions: Analysis of GR chromatin occupancy in human genome substantially contributes to our understanding of the mechanisms and molecular networks underlying the diverse biological effects of glucocorticoids. 2B. Nuclear Receptors and Control of Transcription Abstracts FEBS Journal 275 (Suppl. 1) 99–437 (2008) a 2008 The Authors Journal compilation a 2008 FEBS 139 PP2B-21 CD40 ligation induces immunoproteasome gene expression via the co-ordinated action of NF-jB and of NF-jB mediated de novo synthesis of IRF1 A. Moschonas, M. Kouraki, P. Knox, E. Thymiakou, D. Kardassis and A. G. Eliopoulos Molecular and Cellular Biology Laboratory, School of Medicine, University of Crete, Heraklion, GREECE, Laboratory of Biochemistry, School of Medicine, University of Crete, Heraklion, GREECE Interferon regulatory factors (IRFs) comprise a family of pleiotropic transcription factors which influence development, immune homeostasis and disease pathogenesis. In this study, we demonstrate that stimulation of CD40, a TNF receptor family member with a pivotal role in adaptive and innate immunity, rapidly induces the expression of IRF-1 in carcinoma cells. Using a combination of small molecule kinase inhibitors, RNA interference and CD40 mutagenesis approaches, we show that p65 (RelA) NF-jB but not MAPK signaling is required for the CD40 ligand-induced up-regulation of IRF-1. Chromatin immunoprecipitation and gel-shift assays demonstrated recruitment of p65 NF-jB to the IRF-1 promoter. When fused to a luciferase reporter gene, the IRF-1 promoter responds to CD40 stimulation whereas mutations which abolish NF-jB binding render it unresponsive. Evaluation of NF-jB pathway components suggests the involvement of TAK1, IKKb and IjBa in IRF-1 promoter regulation. NF-jB and de novo synthesized IRF-1 converge to regulate immunoproteasome gene expression, as evident by the recruitment of both transcription factors to the promoter regions of transporter for antigen processing (TAP)-1, TAP-2, tapasin and the low molecular mass polypeptide (LMP)-2 and LMP10. Moreover, the RNA interference-mediated knock-down of IRF-1 reduced, whereas inhibition of NF-jB abolished the effects of CD40 on TAP-1 up-regulation. Collectively, these data reveal a novel mechanism of IRF-1 induction by CD40 which ensures that IRF-1 functions concurrently with NF-jB to facilitate immunoproteasome gene transcription. PP2B-22 Inhibition of the ubiquitous transcription factor Yin Yang 1 by phytosteryl ferulates and their therapeutic potentials R. Nagasaka, K. Ohara and H. Ushio Tokyo University of Marine Science and Technology, Tokyo, JAPAN Rice bran oil accepted worldwide contains a lot of phytosteryl ferulates, one of hydroxycinnamic acid derivatives ubiquitously found in plants, compared with other plant oil. We have reported that phytosteryl ferulates inihibited DNA-binding of NF-jB. In this study, we evaluated the effects of phytosteryl ferulates on DNA binding activities of over 300 transcription factors in RAW 264.7 macrophages. It suggested that phytosteryl ferulates would inhibit one ubiquitous transcription factor Yin Yang 1 (YY1) activation. The transcription factor YY1 is known to have a fundamental role in normal biologic processes such as embryogenesis, differentiation, replication, and cellular proliferation. YY1 exerts its effects on genes involved in these processes via its ability to initiate, activate, or repress transcription depending upon the context in which it binds. It is also generally accepted that the transcription factor acts as an initiator of tumorigenesis. Thus, phytosteryl ferulates might prevent metabolic and immunological diseases by NFjB inhibition and cancer by YY1 repression. The potential clinical significance of the functions will be discussed in this paper, in special the regulation of and the resistance to metabolism and cancer, subsuming complicated cell-cell interactions. PP2B-23 The effects of phytosteryl ferulates on multimeric form of adiponectin secreted from 3T3-L1 adipocytes K. Ohara, R. Nagasaka and H. Ushio Tokyo University of Marine Science and Technology, Tokyo, JAPAN Adiponectin has been postulated to play an important role in the modulation of glucose and lipid metabolisms in insulin-sensitive tissues in mammals. Adiponectin secreted from adipocytes and circulating in blood forms a wide range of multimers from trimers and hexamers to high molecular weight (HMW) multimers, and the ratios among them are closely correlated with insulin sensitivity. Rice bran contains a lot of plant sterols and their ferulic acid esters as compared with other plant oil. It is reported that the phytosteryl ferulates have the LDL cholesterol lowering effect in some mammals. We have recently demonstrated that phytosteryl ferulates enhance adiponectin secretion from adipocytes. In this study, we have investigated the effect of phytosteryl ferulates on the adiponectin multimers secreted from 3T3-L1 adipocytes. Mouse 3T3-L1 cells differentiated to adipocytes were treated with phytosteryl ferulates. The culture media was subjected to SDSPAGE under non-reducing, non-heat-denaturing conditions, followed by western blotting with anti-adipon