Oliver Bruhn

Petra Regenhard

Matthias Michalek

Sven Paul

Christoph Gelhaus

Sascha Jung

Georg Thaller

Rainer Podschun

Matthias Leippe

Joachim Grötzinger

Ernst Kalm
email: ekalm@tierzucht.uni-kiel.de

A novel α-defensin of the horse: gene transcription, recombinant expression and characterisation of the structure and function

Keywords: Antimicrobial peptide, defensin, horse, innate immunity Running Title: An α-defensin of the horse

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INTRODUCTION

An important component of the innate immunity next to the complement pathway and phagocytes are antimicrobial peptides [START_REF] Boman | Antibacterial peptides: basic facts and emerging concepts[END_REF][START_REF] Zasloff | Antimicrobial peptides of multicellular organisms[END_REF], which play a pivotal role as key effectors of the immune system and act as endogenous antibiotics [START_REF] Zasloff | Antimicrobial peptides of multicellular organisms[END_REF][START_REF] Ganz | Defensins: antimicrobial peptides of innate immunity[END_REF]. Antimicrobial peptides are synthesised by circulating phagocytic cells, leukocytes and epithelial cells of mucosal tissues. In mammals, defensins are a predominant class of these peptides that exhibit a direct activity against a broad range of bacteria [START_REF] Ganz | Defensins. Natural peptide antibiotics of human neutrophils[END_REF], fungi [START_REF] Selsted | Activity of rabbit leukocyte peptides against Candida albicans[END_REF] and enveloped viruses [START_REF] Daher | Direct inactivation of viruses by human granulocyte defensins[END_REF].

Defensins are cationic and cysteine-rich peptides with molecular masses ranging from 3 to 5 kDa. They contain six highly conserved cysteine residues forming characteristic intramolecular disulfide bonds. The disulfide array is specific for the three defensin sub-families in mammals: α-, β-, and θdefensins [START_REF] Yang | Mammalian defensins in immunity: more than just microbicidal[END_REF]. An α-defensin gene expression is confirmed only in primates and glires to date. They were found initially in human neutrophil granulocytes, and identified as natural peptide antibiotics [START_REF] Ganz | Defensins. Natural peptide antibiotics of human neutrophils[END_REF].

Further studies showed a gene expression also in mice [START_REF] Eisenhauer | Cryptdins: antimicrobial defensins of the murine small intestine[END_REF], rhesus macaques [START_REF] Tang | Isolation, characterization, cDNA cloning, and antimicrobial properties of two distinct subfamilies of alpha-defensins from rhesus macaque leukocytes[END_REF], rats [START_REF] Yount | Rat neutrophil defensins. Precursor structures and expression during neutrophilic myelopoiesis[END_REF], rabbits [START_REF] Sinha | NP-1, a rabbit alphadefensin, prevents the entry and intercellular spread of herpes simplex virus type 2[END_REF], guinea pigs [START_REF] Selsted | Purification, primary structure, and antimicrobial activities of a guinea pig neutrophil defensin[END_REF], and hamsters [START_REF] Mak | Isolation, antimicrobial activities, and primary structures of hamster neutrophil defensins[END_REF]. Additionally, α-defensin synthesis is presumably unique for a few tissues. The peptides were found in neutrophils, in epithelial cells of the human female urogenital tract [START_REF] Quayle | Gene expression, immunolocalization, and secretion of human defensin-5 in human female reproductive tract[END_REF], in the kidney of rabbits [START_REF] Wu | RK-2: a novel rabbit kidney defensin and its implications for renal host defense[END_REF], and in the Paneth cells of different species [START_REF] Ayabe | Secretion of microbicidal alpha-defensins by intestinal Paneth cells in response to bacteria[END_REF].

Paneth cells are secretory epithelial cells, which are most abundant as specialised ileal cells in the distal small intestine at the base of the crypts of Lieberkuhn. Human Paneth cells synthesise two different α-defensins, HD5 (DEFA5) and HD6 (DEFA6) [START_REF] Jones | Paneth cells of the human small intestine express an antimicrobial peptide gene[END_REF][START_REF] Jones | Defensin-6 mRNA in human Paneth cells: implications for antimicrobial peptides in host defense of the human bowel[END_REF] in addition to the neutrophil human α-defensin peptides 1 to 4 (HNP-1, -2, -3, -4). All α-defensins are synthesised in vivo as inactive precursor proteins. A proteolytic excision of the N-terminal inhibitory anionic propeptide is required for maturation and activation. The signal peptides and propeptides are necessary for correct subcellular sorting and trafficking of the defensins [START_REF] Liu | The pro region of human neutrophil defensin contains a motif that is essential for normal subcellular sorting[END_REF][START_REF] Valore | Intramolecular inhibition of human defensin HNP-1 by its propiece[END_REF], and the propeptide is thought to be the major contributing factor that inhibits the antimicrobial activity by neutralisation of cationic charges of the mature peptide [START_REF] Valore | Intramolecular inhibition of human defensin HNP-1 by its propiece[END_REF].

The active mature α-defensin peptides consist of 29-35 amino-acid residues with a molecular mass of 3-4 kDa. The primary structure shows highly conserved residues, which are indispensable for the Biochemical Journal Immediate Publication. Published on 10 Jul 2007 as manuscript BJ20070747 structural stability. Among them are six invariant cysteines, necessary for the typical α-defensin intramolecular disulfide-bond connectivity (Cys 1 -Cys 6 , Cys 2 -Cys 4 and Cys 3 -Cys 5 ); two charged amino-acid residues Arg 5 , and Glu 13 , forming a conserved salt bridge [START_REF] Wu | Why is the Arg5-Glu13 salt bridge conserved in mammalian alpha-defensins?[END_REF], and Gly 17 , which constitutes a signature structural motif essential for correct folding [START_REF] Xie | Reconstruction of the conserved beta-bulge in mammalian defensins using Damino acids[END_REF]. The tertiary structure is a triple-stranded β-sheet with a beta hairpin that contains cationic amino-acid residues [START_REF] Bals | Antimicrobial peptides and peptide antibiotics[END_REF].

The antimicrobial activity of α-defensins was shown against the gram-negative bacteria Escherichia coli, Salmonella typhimurium, and Enterobacter aerogenes, the gram-positive bacteria Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, the fungal pathogens Candida albicans, and Cryptococcus neoformans, and against the protist Giardia lamblia [START_REF] Tang | Isolation, characterization, cDNA cloning, and antimicrobial properties of two distinct subfamilies of alpha-defensins from rhesus macaque leukocytes[END_REF][24][START_REF] Ouellette | Characterization of luminal paneth cell alpha-defensins in mouse small intestine. Attenuated antimicrobial activities of peptides with truncated amino termini[END_REF][START_REF] Edgerton | Salivary histatin 5 and human neutrophil defensin 1 kill Candida albicans via shared pathways[END_REF][START_REF] Aley | Killing of Giardia lamblia by cryptdins and cationic neutrophil peptides[END_REF][START_REF] Zhang | Contribution of human alpha-defensin 1, 2, and 3 to the anti-HIV-1 activity of CD8 antiviral factor[END_REF].

Furthermore, antiviral activity, particularly an anti-HIV-1 activity was observed [START_REF] Deleeuw | Structure-dependent functional properties of human defensin 5[END_REF]. Defensins are thought to kill bacteria by an initial electrostatic interaction with the negatively charged phospholipids of the microbial cytoplasmatic membrane, followed by membrane permeabilisation [START_REF] Epand | Diversity of antimicrobial peptides and their mechanisms of action[END_REF][START_REF] Sitaram | Interaction of antimicrobial peptides with biological and model membranes: structural and charge requirements for activity[END_REF] and lysis of the microbes [START_REF] Lehrer | Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity[END_REF]. An amphipathic character of the peptide is essential for inserting into the membrane [START_REF] Lehrer | Defensins of vertebrate animals[END_REF].

In this study, we report the transcription of an equine α-defensin gene in the intestine. The peptide was recombinantly expressed in E. coli and its biological activity against various microorganisms was determined. In particular, the mechanism of killing was examined by monitoring membranepermeabilising activity of the equine defensin against viable bacteria and liposomes.

EXPERIMENTAL

Transcriptional analysis

Based on sequence informations of the equine BAC-clone CHORI 241-245H5, Acc.no. AY170305 [START_REF] Looft | Sequence analysis of a 212 kb defensin gene cluster on ECA 27q17[END_REF], primers were constructed to amplify a cDNA representing the potential α-defensin. Total RNA was isolated from 13 epithelial tissues (skin, teat, vagina, tongue, gingiva, esophagus, trachea, lung, small intestine, large intestine, umbilical cord, uterus, meninges) and blood (RNeasy Mini-Kit, QIAGEN, Hilden, Germany). The cDNA was generated by performing RT-PCR using a oligo-dTprimer (OligoT-Bio), 5´-ACT CTA TGA GAA TTC GAT GAG CGA TCT GTT T (n=21) TTV. PCR reactions were performed using the forward primer (U130_L1), 5´-ATC TCC TTT GCA GGG GAT GAA C paired with the reverse primer (L282_L1), 5´-ACG ACA GCA GAG CCT GTA AAT GA and the cDNA as template. The reaction mixtures were incubated at 94 °C for 3 min, followed by 33 cycles at 60 °C for 50 s, 72 °C for 50 s, and 94 °C for 1 min. Finally, agarose gel electrophoresis was performed to determine the transcription.

Molecular cloning and sequencing of DEFA1

The generated product of the small intestine was extracted from the gel with the Gel-Extraction Kit (QIAGEN) and purified with the Reaction Cleanup Kit (QIAGEN). The sequences were determined (DLMBC, Berlin, Germany) by using the forward primer and reverse primer previously used for expression analysis. The resulting sequences revealed sequence overlays (data not shown). New primers were generated, the forward primer (DEFA5L_up1), 5´-TTG ACT CCC AGC CAT GAG (coding sequence is underlined) and the reverse primer (TailPrimer3´), 5´-ACT CTA TGA GAA TTC GAT GAG CGA TCT G (complementary to the OligoT-Bio primer) to determine the complete cDNA sequence. A 3´-RACE [START_REF] Schramm | A simple and reliable 5'-RACE approach[END_REF] was performed with cDNA from the small intestine as template under the following PCR conditions: 94 °C for 3 min, followed by 33 cycles at 58 °C for 50 s, 65 °C for 45 s and 94 °C for 1 min. The amplified product was purified by gel extraction (QIAGEN). For sequencing, 68 ng of the purified product was subcloned in the pDRIVE cloning vector according to the manufacturer´s specifications (Cloning-Kit, QIAGEN). A test PCR with the forward primer (DEFA5L_up1) and the reverse primer (DEFA5L_lo300), 5´-ACG ACA GCA GAG CCT GTA AAT G was performed with 20 of the grown clones as a template. Positive clones were sequenced (DLMBC, Berlin, Germany). The obtained sequences were aligned (ClustalW, www.ebi.ac.uk/clustalw/) and analysed.

Preparation of the cDNA for recombinant peptide expression

The cDNA coding for the mature petide of the α-defensin, corresponding to nucleotides 193 to 296 of the equine α-defensin cDNA (Acc. no. EF379126) was amplified using the forward primer (DEA6_mat_U2), 5´-GGT ATT GAG GGT CGC TCC TGC ACC TGC AGA CGT, paired with the reverse primer (DEA6_mat_L1), 5´-AGA GGA GAG TTA GAG CC TCA GCG ACG ACA GCA GAG (peptide specific sequence is underlined) and the pDRIVE cloning vector containing the complete cDNA as template. The reaction was performed by incubating the reaction mixture at 94 °C for 3 min, followed by 33 cycles at 61 °C for 30 s, 68 °C for 1 min and 94 °C for 45 s. The sample was purified by gel extraction. The purified PCR products (0.2 pM) were treated 30 min at 22 °C with 0.16 U T4 DNA Polymerase (QIAGEN) and 3 mM dGTP to generate the overhangs for the annealing reaction. The product was ligated into the vector pET-30 Xa/LIC (NOVAGEN, Darmstadt, Germany).

Recombinant expression of DEFA1

The vector was transformed into the E. coli-strain BL21 (DE3) by heat-shock (90 s at 42 °C). The cells were grown at 37 °C to an OD 600 of 0.5 in 1.5 l Luria-Bertani (LB)-Medium (ROTH, Karlsruhe, Germany) containing 30 µg/ml kanamycin [START_REF] Hristova | Structure, location, and lipid perturbations of melittin at the membrane interface[END_REF]. The expression of the fusion petide was induced with 1 mM IPTG (isopropyl-D-1-thiogalactopyranoside) followed by an incubation for 4 h at 37 °C. Bacterial cells were harvested by centrifugation at 6000 x g for 20 min at 4 °C and lysed by resuspending the bacterial cell pellet in 40 ml 6 M guanidine HCl in 100 mM Tris-HCl, pH 8.0 followed by sonication at 70% power for 3 min (BANDELIN Sonopuls, Berlin, Germany). The lysate was clarified by centrifugation at 30,000 x g for 30 min at 4 °C, supernatant was sterilised using a membrane filter with a pore-size of 0.45 µm (SARTORIUS, Göttingen, Germany) prior to peptide purification.

Purification of the recombinant peptide

The His 6 -tagged α-defensin fusion peptide was purified by performing nickel-nitrilotriacetic acid metal-affinity chromatography using prepacked Ni-NTA superflow columns (QIAGEN). Clarified lysates were transferred to the column and drained by gravity flow. Washing was performed with 6 M guanidine HCl in 100 mM Tris-HCl, pH 8.0 including 15 mM imidazole, and the His 6 -tagged peptide was eluted with 6 M guanidine HCl in 100 mM Tris-HCl, pH 8.0 including 750 mM imidazole. The samples were stored at -20 °C.

The buffer of the dissolved fusion peptides was exchanged by using desalting columns PD-10 (GE HEALTHCARE, Uppsala, Sweden, bed volume 8.3 ml). Ni-NTA purified peptides were transmitted to a PD-10 column and eluted with cleavage buffer consisting of 20 mM Tris-HCl, 50 mM NaCl, 1 mM CaCl 2 , pH 6.5. The buffer exchange procedure was repeated twice. The eluted His 6 -tagged peptide was quantified photometrically by measuring the UV absorpion at 280 nm based on the extinction coefficient [START_REF] Waxman | Determination of the tryptophan:tyrosine ratio in proteins[END_REF].

The fusion peptide was cleaved for 12 h at room temperature with 10 U Factor Xa protease (QIAGEN) per 150 µg peptide in cleavage buffer.

Reversed-phase chromatography

After cleavage, the mature peptide was purified immediately to apparent homogeneity by using reversed-phase high performance liquid chromatography (RP-HPLC). The sample was applied to a C-18 preparative RP-HPLC VP 250/10 Nucleosil 300-7 column (MACHEREY-NAGEL, Düren, Germany), and the peptide was eluted after 16 min using a 22-40% acetonitrile gradient developed over 90 min. Finally, the peptide was lyophilised, and redissolved in 0.01% trifluoroacetic acid (TFA) for storage at -20 °C and further studies.

Circular dicroism (CD spectroscopy)

CD measurements were carried out on a JASCO J-720 spectropolarimeter (JAPAN SPECTROSCOPIC CO., Ltd., Tokyo, Japan) calibrated according to reference [START_REF] Chen | Two-point calibration of circular dichrometer with d-10camphorsulfonic acid[END_REF]. The spectral bandwidth was 2 nm. Three scans were recorded between 190 nm and 250 nm with 1 nm acquisition steps and the spectrum was baseline corrected. Measurements were carried out at room temperature and cuvettes of 1.0 mm optical path length were used. The peptide concentration was 15 µM in 0.01% TFA.

Mass spectrometry

The molecular identity and homogeneity of the preparation was verified by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The samples were mixed with a saturated matrix solution of sinapinic acid prepared in 80% acetonitrile/0.1% TFA and spotted on a stainless steel sample target. Mass spectra were obtained on a MALDI-TOF/TOF mass spectrometer (4700 Proteomics Analyser, Applied Biosystems, Framingham, MA, USA) in the positive ion linear mode and calibrated externally. The average mass of the proteins were compared to the theoretical average mass of DEFA1 considering potential disulfide formation.

Antimicrobial activity

Following bacterial strains were used in a radial diffusion assay as described previously [START_REF] Lehrer | Ultrasensitive assays for endogenous antimicrobial polypeptides[END_REF] to mid-log phase in trypticase soy broth (TSB), suspended into agar (1% agarose in 10 mM sodium phosphate pH 7.4 supplemented with a 1:100 dilution of 1 x TSB, and poured into petri dishes (10 ml). After hardening, 5 µl of peptide test solution was pipetted into wells formed with a biopsy punch (diameter: 3 mm) and allowed to incubate at 37 °C overnight. Lysozyme was used as a control peptide, 0.01% TFA served as negative control. Plates were then overlayed with 10 ml of 1% agar supplemented with double strength TSB (caseine peptone 3.4%, tryptic soy peptone 0.6%, glucose 0.5%, NaCl 1%, and K 2 HPO 4 0.5%). After incubation for 3 h, zones of growth inhibition surrounding the wells were measured.

Additionally, LD 90 and MBC (minimal bactericidal concentration) values of the peptide (≥ 90% or ≥ 99.9% killing of the target organisms) were determined by using the test strain pattern described for the radial diffusion assay with the exception of E. coli ATCC 11030. In addition, gram-negative Klebsiella pneumoniae ATCC 13883, Enterobacter cloacae ATCC 13047, P. aeruginosa ATCC 11440, gram-positive Enterococcus faecalis ATCC 29212, Ec. faecalis PEG 205 (wild type), and Streptococcus pyogenes ATCC 12344 were introduced. Briefly, after a 2-3 h growth period in brain heart infusion broth at 36 ± 1 °C, the bacteria were washed three times in 10 mM sodium phosphate buffer (pH 7.4) and adjusted to 10 4 -10 5 colony forming units (cfu) per ml in the same buffer. Ten µl of peptide solution (range of final concentration tested: 0.05-6.1 µM) were added to 100 µl of the bacterial suspension and incubated at 36 ± 1 °C for 2 h before colony forming units were determined.

The peptide solvent (0.01% TFA) served as negative control. Permeabilisation of the bacterial cytoplasmic membrane allows the dye to cross this membrane and to intercalate with the DNA. When excitated at 495 nm, the binding of the dye to DNA resulted in an increase of emitted fluorescence at 538 nm, which was measured in a microtiter plate reader (Fluoroskan II; Labsystems). Membrane-permeabilising activity of the peptides was expressed as percentage of permeabilised bacteria. For monitoring the activity at pH 5.2, 20 mM MES, 25 mM NaCl was used. As a control antimicrobial peptide, cecropin P1 (in HPLC-grade; SIGMA-ALDRICH) was used in parallel. For maximum permeabilisation of the bacteria (100% value), cells were incubated with 70% ethanol for 10 min. The values were expressed as the mean of two independent experiments, each performed in duplicates.

Permeabilisation of bacterial membranes

Assay for pore-forming activity

Pore-forming activity of samples was determined by measuring fluorimetrically the dissipation of a valinomycin-induced membrane potential in liposomes prepared from crude soy bean phospholipids [START_REF] Leippe | Pore-forming peptide of pathogenic Entamoeba histolytica[END_REF]. Fluorescence was measured by a fluorescence spectrophotometer (model LS 50B; PERKIN ELMER) using excitation and emission wavelengths of 620 nm and 670 nm, respectively. Pore-forming activity was measured as the initial change in fluorescence intensity over time after adding the sample. As a control pore-forming peptide, alamethicin (in HPLC-grade; SIGMA-ALDRICH) was used in parallel.

Molecular modelling

The three-dimensional model for the equine α-defensin was generated using the X-ray structure of the human α-defensin HNP-3 (PDB acc. code: 1dfn) as a template. According to the alignment, aminoacid residues were exchanged in the template structure. Insertions and deletions were modelled by using a database search approach included in the software package WHAT IF [START_REF] Vriend | WHAT IF: a molecular modeling and drug design program[END_REF]. The database was searched for a peptide sequence of the appropriate length, which was fitted into the template. All loops were selected from the database so as to give a minimum root mean square distance between the ends of the loops and helices. Loops with unfavourable backbone dihedral angles or van der Waals clashes were excluded. Finally, these model structures were energy minimised, using the steepest descent algorithm implemented in the GROMOS force field [START_REF] Soares | An improved nucleic acid parameter set for the GROMOS force field[END_REF]. The structural representation was performed with the RIBBON programme [START_REF] Kraulis | A program to produce both detailed and schematic plots of protein structures[END_REF].

RESULTS

Transcriptional analysis

An amplified product could solely be verified in the small intestine of the horse (FIG. 1). This analysis failed to detect gene transcription in skin, teat, vagina, tongue, gingiva, esophagus, trachea, lung, large intestine, umbilical cord, uterus, meninges and blood. The amplified product of the horse small intestine was approximately 340 base pairs long. The specific transcription of the equine α-defensinlike gene suggested the existence of an α-defensin in the intestine of the horse.

Determination of the primary structure of the DEFA1 precursor

Sequence overlays were determined by sequencing the amplified product from the gut with primers used for transcriptional analysis. It could not be excluded that more than one product has been amplified. Therefore, subcloning was performed with amplified products generated by 3´-RACE to determine the complete coding sequence of the amplified product. According to the transcriptional analysis, cDNA of the small intestine was used for the PCR reaction. Positive clones were sequenced with forward and reverse primers used for 3´-RACE. Two distinct cDNA sequences, DEFA5L (already known from the BAC clone CHORI 241-245H5), and a new sequence, DEFA1 (Acc. no. EF379126) were identified.

The cDNA sequence of the DEFA1 gene showed a open reading frame of 297 nucleotides including start and stop codon (FIG. 2 A). In comparison with sequences available in public databases, the region of the putative propetide and mature peptide was predicted. The putative coding region of both, the signal peptide and the propeptide is 192 nucleotides in length, the coding region of the mature peptide consists of 105 nucleotides. Alignment (ClustalW) of the DEFA1 amino acid sequence with the equine DEFA5L sequence (CAJ01789) and sequences of the human DEFA5 (EAW80489), DEFA6 (AAC50382) and HNP-3 (NP_005208), chimpanzee DEFA5 (NP_0010112657), rhesus macaque α-defensin 5 (AAW51369), rat DEFA5 (P82106), α-defensin 6 (AAT91348), and NP-3 (Q62713) showed a relative conserved region of the signal peptide and a more divergent region of the mature peptide (FIG. 2 B). The highest identity was found to equine DEFA5L with 84% amino acid sequence identity and to the human and chimpanzee DEFA5 as well as to the rat neutrophil peptide (NP-3) with 48% sequence identity to each. The rhesus macaque α-defensin 5 showed an identity of 44% to DEFA1, the rat Paneth cell specific α-defensins DEFA5 and defensin 6 an identity of 39%, human DEFA6 of 37%, whereas the lowest identity was found to human HNP-3 (35%). The six cysteine residues (Cys 2 , Cys 4 , Cys 10 , Cys 20 , Cys 31 and Cys 32 ) are completely conserved in the peptides.

Equally, the arginine and glutamine residues (Arg 5 and Glu 13 ), necessary for the intramolecular salt bridge, are conserved as well as the glycine residue at position 17, essential for correct folding. The 34 amino-acid residues long putative mature peptide starts with a serine residue followed by the first cysteine. Including the signal peptide and propeptide the complete primary structure of the DEFA1 precursor consists of 98 amino-acid residues.

Recombinant expression and purification of DEFA1

Expression of recombinant DEFA1 was performed by cloning the cDNA sequence of the mature αdefensin peptide (FIG. 2 A) into the pET30-Xa/LIC vector and transfecting E. coli BL21 (DE3) with the vector. The fusion peptide was exclusively synthesised in inclusion bodies and therefore was only detected in the pellet of cells after cell lysis and centrifugation at 30,000 x g (FIG. 3 A). Analysis of bacterial extracts by SDS-PAGE at different time points revealed a peptide synthesis in inclusion bodies already during the first 30 min after induction of gene expression. A further increase of peptide concentration after induction was observed after an expression time of up to four hours (FIG. 3 B).

The recombinant product, confirmed by SDS-PAGE, had a molecular mass of approximately 10 kDa closely to the theoretical mass of 9.046 kDa. After nickel-nitrilotriacetic acid metal-affinity chromatography, the cleavage of the product with Factor Xa resulted in a peptide of approximately 5 kDa, determined by Tricine-PAGE (FIG. 3 C). This result is in good agreement with the expected mass of 4.071 kDa.

The recombinant DEFA1 was purified to homogeneity by performing RP-HPLC. Three conspicuous peaks appeared at elution times of 13.4, 14.0, and 15.7 min (FIG. 4 A). The fractions were analysed by MALDI-TOF mass spectrometry. The fraction F1 showed an unknown peptide mixture with peptide masses of approximately 4090, 5003 and 5035 Da. Fractions F2 and F3 contained peptides with the mass of 4071.8 Da (FIG. 4 B). Notably, for the mature DEFA1 peptide an average mass of 4071.8 Da was calculated, provided that the six cysteine residues are involved in disufid bonds. Whereas in fraction F2 the His 6 -tag (4.987 kDa) was eluted in addition to the mature peptide in fraction F3 homogeneous material representing the DEFA1 peptide was found. The accurate monoisotopic mass of the peptide was also determined (4068.9 Da) and confirmed the molecular identity of DEFA1. F3 eluates were used for further studies. The eluates were lyophilised and the purified peptide was resuspended in 0.01% TFA for further investigations. Finally, MALDI-TOF mass spectrometry was performed to proof the purity of the DEFA1 peptide (data not shown).

Structural characterisation

The recombinantly expressed DEFA1 was structurally characterised by CD spectroscopy. The CD spectrum (in the range of 190 nm and 250 nm) is shown in FIG. 5 and displays a minimum at 203 nm.

The shape of the spectrum is indicative for a β-sheet structure with a high content of P 2 elements [START_REF] Sreerama | Structural composition of βIand βII-proteins[END_REF]. This is in agreement with the known α-defensin structures.

Antimicrobial activity of DEFA1

To assess the antimicrobial activity of DEFA1, a radial diffusion assay was performed at first. As shown in FIG. 6, treatment of microorganisms with the recombinant equine α-defensin resulted in a visible inhibition of the growth (zones of clearing) against all of the target organisms with the exception of B. cepacia (data not shown). The relative potential of the peptide varied depending on the microorganisms tested. Whereas DEFA1 was only lightly active against P. aeruginosa ATCC 10145, potent activity was found against Bac. megaterium, all E. coli-strains, Staph. epidermidis and Staph. aureus-strains. Against seven test strains (all E. coli-strains, Staph. epidermidis and Candida albicans), peptide concentrations as low as 4.7 µM led to growth inhibition zones. The Staph. aureus strains were inhibited at concentrations of ≥ 9.3 µM, Candida albicans was susceptible at ≥ 18.7 µM.

Additionally, the minimal concentration of the equine DEFA1 that killed 90% (LD 90 ) and 99.9% (MBC) of 17 target microorganisms is shown in TABLE 1. All organisms were tested with various defensin concentrations (0.05 to 6.1 µM). This range was sufficient to determine the lethal peptide dose (LDs) of the target organisms. DEFA1 displays a broad spectrum of antimicrobial activity against fermenting and non-fermenting gram-negative strains, against gram-positive cocci (genera Staphylococcus, Streptococcus, and Enterococcus), spore-forming Bac. megaterium, and the yeast C. albicans. Bac. megaterium was more susceptible to DEFA1 than any of the other strains tested. In general, E. coli strains were more susceptible to the peptide than other fermenting gram-negatives such as K. pneumoniae and Eb. cloacae, which are weakly susceptible to the peptide. Non-fermenting gram-negative bacteria such as P. aeruginosa and B. cepacia were only weakly susceptible or showed no sensitivity to DEFA1. The gram-positive cocci such as Staph. aureus, Staph. epidermidis, Ec. faecalis, and Strep. pyogenes exhibited a high sensitivity to DEFA1 at reliable peptide concentrations.

The yeast Candida albicans showed a MBC value of 0.8 µM, B. cepacia was not killed up to peptide concentrations of 6.1 µM. These results correspond with the results from the radial diffusion assay.

Characterisation of the peptide's mode of action

To investigate the mode of action of DEFA1, we used Bac. megaterium and the fluorescent dye SYTOX Green. As soon as the dye can enter the bacterial cell it intercalates with the DNA and its fluorescence will be enhanced. This assay confirmed that DEFA1 is potently active against these Gram-positives (FIG. 7 A). Notably, it demonstrated that the peptide rapidly permeabilises bacterial membranes. As DEFA1 contains a histidine residue in its primary structure, we evaluated the effect of a more acidic pH at which the histidine residue is protonated on the membrane-permeabilising activity. The efficacy of the horse defensin to induce membrane lesions in viable bacteria was found to be very similar at pH 5.2 and pH 7.4 and comparable to that of the well-known antimicrobial peptide cecropin P1 (FIG. 7 B).

We also measured the pore-forming activity of DEFA1 to further characterise its mode of action by using a minimalistic membrane system. More precisely, we monitored the dissipation of a membrane potential induced in liposomes composed of azolectin, a crude phospholipid mixture from soy bean. It became evident that the equine defensin displayed pore-forming activity (FIG. 8), although only moderately when compared to alamethicin, the prototype of a pore-forming peptide acting according to the barrel-stave model [START_REF] Baumann | A molecular model of membrane excitability[END_REF].

DISCUSSION

The existence of an α-defensin peptide in the small intestine of the horse was indicated by transcriptional analysis, and the obtained amino-acid sequence aligned to other Paneth cell specific αdefensin sequences. Interestingly, an α-defensin expression in other species next to primates and rodents was not reported to date. The horse is now added to the group of animals expressing αdefensins, and this may lead to a new perception in the evolution of defensins. A possible ancestral gene for DEFA5 may already have been present before euarchontoglires (including glires and primates) and laurasiatheria (including perissodactyla amongst others) evolved to two different subgroups. It is unlikely that the identified equine DEFA1 has developed independently as indicated by the high identity of the amino acid sequences between the equine DEFA1 and other known αdefensins (FIG. 2 B). This is supported by the finding of genomic α-defensin like sequences in basal mammals [START_REF] Lynn | Discovery of α-defensins in basal mammals[END_REF]. DEFA1 revealed the typical conservation of a signal-and negatively charged prosegment, and the αdefensin characteristic conserved amino-acid residues, including the six cysteines, Arg 5 , Glu 13 and Gly 17 . Most α-defensins contain no or only one charged or polar uncharged amino-acid residue following the last two cysteines. C-terminal tails, rich in charged or polar uncharged amino-acid residues are predicted to be exposed to the surface to influence the overall charge and amphipathicity of the peptide. The equine defensins DEFA1 and DEFA5L have two C-terminal Arg residues (FIG. 2 B). An extended C-terminus is also shown on rhesus macaque α-defensin 5. In previous studies, a general relationship between higher defensin cationicity and a greater microbicidal potency was noted [START_REF] Selsted | Activity of rabbit leukocyte peptides against Candida albicans[END_REF]. It was supposed that the net charge covers a different antimicrobial spectrum and efficiency of these peptides and is strongly correlated with antimicrobial activity of the defensins [START_REF] Raj | Large-scale synthesis and functional elements for the antimicrobial activity of defensins[END_REF]. Equine DEFA1 is less cationic (+ 2) than human DEFA5 (+ 5), and Paneth cell α-defensins from rhesus macaque small intestine (+ 4.5 to + 7.5), but equine DEFA1 showed at least similar antimicrobial activities. Accordingly, the relative cationicity did not appear to be a major factor in the killing of bacteria. This result is consistent with those of previous studies demonstrating that the overall microbicidal activity of α-defensins are not merely a function of their net charge [START_REF] Cullor | Bactericidal potency and mechanistic specificity of neutrophil defensins against bovine mastitis pathogens[END_REF], but may influence a strain specifity. Collectively, despite the broad antimicrobial spectrum of DEFA1, the most sensitive targets of this peptide are gram-positive bacteria. It appears that a preference for particular bacteria applies for both, human DEFA5 and equine DEFA1.

The mode of action of Paneth cell α-defensins was not studied until date. For antimicrobial peptides in general a two-step mechanism is proposed to explain their killing effect on microorganisms. Most cationic peptides bind to the target cell surface due to their content of negatively charged phospholipids or other moieties. Many of them subsequently disturb the membrane integrity [START_REF] Zasloff | Antimicrobial peptides of multicellular organisms[END_REF].

Likewise, killing of bacteria by the equine defensin was accompanied with permeabilisation of the bacterial cytoplasmic membranes similar to the action of other antimicrobial peptides such as cecropin P1. Moreover, in a minimalistic system using the depolarisation of liposomes as a measure of poreforming activity it became evident that DEFA1 is moderately active in creating pores in artificial membranes, a property described previously for human alpha-defensins [START_REF] Wimley | Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores[END_REF].

In recent studies, a specific deficiency of human DEFA5 was observed in patients with ileal Crohn´s disease, a chronic disease of the intestine characterised by inflammation of the gut. The horse shows similar disease patterns, for example anterior enteritis (duodenitis) or colitis X, catarrhalhaemorrhagic inflammations of the gut. It will be interesting to examine a possible relationship of these infectious diseases with a possible deficiency of equine Paneth cell α-defensins.

FIGURE LEGENDS

  Bacteria with compromised membranes were detected by monitoring the fluorescence of the DNAbinding dye SYTOX Green (INVITROGEN, Molecular Probes). Bac. megaterium ATCC 14581 in mid-logarithmic phase was washed twice with and resuspended in 10 mM HEPES, pH 7.4 containing 25 mM NaCl. A flat bottom 96-well microtiter plate (SARSTEDT, Germany) was coated with 0.1% bovine serum albumin (A2153, SIGMA-ALDRICH, Deisenhofen, Germany) for 15 min prior to its use in the assay. Peptides were twofold serially diluted in 10 mM HEPES, 25 mM NaCl, pH 7.4. Bacteria (1 x 10 5 CFU/50 µl) were incubated with the diluted peptides (25 µl) and 2 µM of the fluorescent dye SYTOX Green (25 µl; in 10 mM HEPES, 25 mM NaCl, pH 7.4) at 20 °C for 1 h.

A

  three-dimensional model of the DEFA1 structure is shown in FIG. 9. The model displays a small βsheet consisting of three β-strands flanked by large unstructured regions. The long loop connecting the first and the second β-strand contains several cationic amino-acid residues. Together with the two arginines located at the C-terminus, this loop forms an epitope with a high positive charge density. Alignment of DEFA1 and DEFA5L with other amino-acid sequences revealed a homology to human DEFA5. The antimicrobial properties of human DEFA5 have been investigated comprehensively [24]. In summary, the authors established that human DEFA5 efficiently kills gram-negative and gram-positive bacteria at comparable peptide concentrations and showed a consistently high activity against a broad spectrum of microorganisms including fungi. Human DEFA5 was potent in almost the same manner as the neutrophil peptides HNP 1-4 against gram-positive Staph. aureus, but displayed a conspicuous higher activity against E. coli. By comparing the activities of equine DEFA1 against E. coli, Staph. aureus and Staph. epidermidis we also determined a higher activity against E. coli. Other strains of gram-negative bacteria such as Eb. cloacae, K. pneumoniae, P. aeruginosa and B. cepacia are weakly sensitive. Similarly, gram-positive cocci such Staph. aureus, Staph. epidermidis, Strep.pyogenes, and Ec. faecalis are highly sensitive to DEFA1 at reliable peptide concentrations.

FIG. 1 .FIG. 2 .FIG. 3 .FIG. 4 .A.FIG. 5 .FIG. 6 .FIG. 7 .FIG. 8 .FIG. 9 .

 123456789 FIG. 1. Transcriptional analysis of DEFA1 in various equine tissues.

Biochemical Journal Immediate Publication. Published on 10 Jul 2007 as manuscript BJ20070747 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  : Gramnegative Escherichia coli ATCC 11303, ATCC 11775, ATCC 25922, ATCC 35218, and D31, Pseudomonas aeruginosa ATCC 10145, Burkholderia cepacia ATCC 25416, and gram-positive Staphylococcus aureus ATCC 6538, and ATCC 12600, Staph. epidermidis ATCC 14990, Bacillus megaterium ATCC 14581, and the yeast Candida albicans ATCC 24433. The organisms were grown

TABLE 1 . LD 90 and MBC values of DEFA1 against different microorganisms.

 1 LD 90 and MBC (minimal bactericidal concentration) values are defined as lethal peptide dose in µM that killed 90% (LD 90 ) or 99.9% (MBC) of the target organisms. The highest peptide concentration tested was 6.1 µM. >, no killing found at the indicated concentration

		Gram stain
	Test organism	reaction LD 90 [µM] of DEFA1 MBC [µM] of DEFA1
	E. coli ATCC 11775	-	0.2	0.4
	E. coli ATCC 25922	-	0.2	0.4
	E. coli ATCC 35218	-	0.05	0.4
	E. coli D31	-	0.4	0.8
	K. pneumoniae ATCC 13883	-	3	>6.1
	Eb. cloacae ATCC 13047	-	6.1	>6.1
	P. aeruginosa ATCC 10145	-	3	>6.1
	P. aeruginosa ATCC 11440	-	1.5	3
	B. cepacia ATCC 25416	-	>6.1	>6.1
	Staph. aureus ATCC 6538	+	0.4	0.8
	Staph. aureus ATCC 12600	+	0.4	1.5
	Staph. epidermidis ATCC 14990	+	0.8	1.5
	Ec. faecalis ATCC 29212	+	0.2	6.1
	Ec. faecalis PEG 205 (wild type)	+	0.4	3
	Strep. pyogenes ATCC 12344	+	0.8	0.8
	Bac. megaterium ATCC 14581	+	0.05	0.2
	C. albicans ATCC 24433		0.8	0.8

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 10 Jul 2007 as manuscript BJ20070747 © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

We thank the BMBF (FUGATO) and the German Research Council (DFG, SFB 617) for supporting this study. Many thanks to Sonja Hollmer from the Institute of Biochemistry and Heidrun Ließegang from the Zoological Institute, University of Kiel for their technical assistance, and to Ulrike Diesterbeck of the Institute of Virology, University of Göttingen for valuable discussions.


Albi Elisabetta

Lazzarini Remo

Viola Magni Mariapia

Elisabetta Albi
email: ealbi@unipg.it

PHOSPHATIDYLCHOLINE/SPHINGOMYELIN METABOLISM CROSSTALK INSIDE THE NUCLEUS

Keywords: DAG, diacylglycerol, PC, phosphatidylcholine, ceramide, chromatin, diacylglycerol, nuclear membrane, sphingomyelin

It is known that the phospholipids represent a "minor component" of chromatin. Recently it has been highlighted that these lipids are metabolised directly inside the nucleus thanks to the presence of the enzymes related to their metabolism such as neutral sphingomyelinase, sphingomyelin-synthase, reverse sphingomyelin-synthase and phosphatidylcholine-specific phospholipase C. The chromatin enzymatic activities change during cell proliferation, differentiation and/or apoptosis independently of the enzyme activities present in the nuclear membrane, microsomes or cell membranes. The present research was aimed to investigate the crosstalk of the lipid metabolisms in nuclear membrane and chromatin isolated from rat liver in vitro and in vivo. The effect of neutral-sphingomyelinase activity on phosphatidylcholinespecific phospholipase C and sphingomyelin-synthase, which enrich the intranuclear diacylglycerol pool and the effect of phosphatidylcholine-specific phospholipase C activity on neutral sphingomyelinase and reverse sphingomyelin-synthase, which enrich the intranuclear ceramide pool, were investigated. The results show that in chromatin exists a phosphatidylcholine/sphingomyelin metabolism crosstalk which regulates the intranuclear ceramide/diacylglycerol pool. Its specificity was demonstrated by D609 inhibitor effect. The chromatin lipid metabolism is activated in vivo during cell proliferation indicating that it could play a role on cell function. The possible mechanism of the crosstalk was discussed considering the recent literature in the field.

Introduction

The involvement of sphingomyelin (SM) and phosphatidylcholine (PC) in signal transduction [START_REF] Levade | Signalling sphingomyelinases: which, where, how and why?[END_REF][START_REF] Wright | Cell membranes and apoptosis: role of cardiolipin, phosphatidylcholine, and anticancer lipid analogues[END_REF] and the role of diacylglycerol (DAG) and ceramides in cell proliferation, differentiation and apoptosis have been widely described [START_REF] Lang | Molecular species analysis of 1,2-diglycerides on phorbol ester stimulation of LA-N-1 neuroblastoma cells during proliferation and differentiation[END_REF][START_REF] Hannun | The Ceramide-centric universe of lipidmediated cell regulation: stress encounters of the lipid kind[END_REF][START_REF] Luberto | Ceramide regulation of apoptosis versus differentiation: a walk on a fine line. Lessons from neurobiology[END_REF]. SM is metabolised by sphingomyelinase (SMase) producing ceramide and phosphocholine (PPC) [START_REF] Spence | Sphingomyelinases[END_REF] and by reverse-sphingomyelin-synthase (RSM-synthase) that synthesises PC using DAG and PPC group of SM, freeing ceramide [START_REF] Marggraf | The phosphorylcholine acceptor in the phosphatidylcholine:ceramide cholinephosphotransferase reaction. Is the enzyme a transferase or a hydrolase?[END_REF]. Phosphatidylcholine-specific phospholipase C (PC-PLC) degrades PC producing DAG and PPC [START_REF] Exton | Phosphatidylcholine breakdown and signal transduction[END_REF] whereas sphingomyelin-synthase (SM-synthase) synthesises SM using ceramide and PPC group of PC, freeing DAG [START_REF] Allan | Resynthesis of sphingomyelin from plasma-membrane phosphatidylcholine in BHK cells treated with Staphylococcus aureus sphingomyelinase[END_REF]. It is known that PC breakdown activates PKC [START_REF] Van Dijk | Diacylglycerol generated by exogenous phospholipase C activates the mitogen-activated protein kinase pathway independent of Ras-and phorbol ester-sensitive protein kinase C: dependence on protein kinase C-zeta[END_REF], a family of serine/threonine kinases which participates in signal transduction of many cells and mediates a wide number of intracellular functions [START_REF] Cross | Serine/threonine protein kinases and apoptosis[END_REF].

On the other hand it has been demonstrated that cellular DAG may activate the SMase with consequent inactivation of PKC [12] whereas the activation of SM-synthase causes an increase of DAG with positive effect on this enzyme [START_REF] Hampton | Sphingomyelin synthase and PKC activation[END_REF]. The co-operation between different lipid pathways has been also reported in cells stimulated with TNF . In fact, TNF acts on NF-kB through an initial activation of PC-PLC with production of DAG which in turn stimulates SMase activity with consequent ceramide formation and specific kinase isoform activation [START_REF] Schutze | TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase Cinduced "acidic" sphingomyelin breakdown[END_REF]. Moreover it has been demonstrated that cellular SMase and SMsynthase are involved in cell proliferation [START_REF] Maupas-Schwalm | The sphingomyelin/ceramide pathway is involved in ERK1/2 phosphorylation, cell proliferation, and uPAR overexpression induced by tissue-type plasminogen activator[END_REF] and apoptosis process [START_REF] Van Der Luit | Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin-and cholesterol-deficiency in lipid rafts[END_REF] respectively.

During the last then years many evidences highlighted the presence of a phospholipids (PLs) fraction in the nucleus that changes in relation to cell function [START_REF] Albi | The role of intranuclear lipids[END_REF][START_REF] Ledeen | Nuclear lipids: key signalling effectors in the nervous system and other tissues[END_REF][START_REF] Ledeen | Sphingolipids of the nucleus and their role in nuclear signaling[END_REF][START_REF] Martelli | Metabolism and signaling activities of nuclear lipids[END_REF]. These lipids change thanks to the existence of neutral-SMase (N-SMase) [START_REF] Watanabe | Increase of nuclear ceramide through caspase-3-dependent regulation of the "sphingomyelin cycle" in Fas-induced apoptosis[END_REF][START_REF] Tamiya-Koizumi | Existence of Mg2+-dependent, neutral sphingomyelinase in nuclei of rat ascites hepatoma cells[END_REF][23][START_REF] Romanenko | RNA-polymerase, DNA-polymerase, DNA-methyltransferase and sphingomyelinase activities in liver nuclei of rats of different Age[END_REF][START_REF] Albi | Chromatin neutral spingomyelinase and its role in hepatic regeneration[END_REF][START_REF] Mizutani | Nuclear localization of neutral sphingomyelinase 1: biochemical and immunocytochemical analyses[END_REF], SM-synthase [START_REF] Albi | Sphingomyelin-Synthase in Rat Liver Nuclear Membrane and Chromatin[END_REF][START_REF] Kavok | Increase in diacylglycerol production by liver and liver cell nuclei at old age[END_REF], PC-PLC [START_REF] Albi | Phosphatidylcholine-Dependent Phospholipase C in Rat Liver Chromatin[END_REF][START_REF] Ramoni | Phosphatidylcholine-specific phospholipase C in mitogen-stimulated fibroblasts[END_REF], and RSM-synthase [START_REF] Albi | Reverse Sphingomyelin-Synthase in Rat Liver Chromatin[END_REF] activity in the nucleus. The nuclear N-SMase activity is involved in cell proliferation [START_REF] Alessenko | Neutral sphingomyelinase: localization in rat liver nuclei and involvement in regeneration/proliferation[END_REF] and/or apoptosis [START_REF] Tsugane | A possible role of nuclear ceramide and sphingosine in hepatocyte apoptosis in rat liver[END_REF]. The purification of the subnuclear fractions has permitted to demonstrate that the enzymes localised in the chromatin have different pH optimum and Km from those present in nuclear membrane [START_REF] Albi | Chromatin neutral spingomyelinase and its role in hepatic regeneration[END_REF][START_REF] Albi | Sphingomyelin-Synthase in Rat Liver Nuclear Membrane and Chromatin[END_REF][START_REF] Albi | Phosphatidylcholine-Dependent Phospholipase C in Rat Liver Chromatin[END_REF][START_REF] Albi | Reverse Sphingomyelin-Synthase in Rat Liver Chromatin[END_REF]. At the moment the nuclear membrane and chromatin enzymes have not been purified and characterised and therefore do not exist specific antibodies that could be used for the study of their exact localization. Nevertheless, an electron microscopy analysis on the rat liver sections performed by using N-SMase conjugated with colloidal gold particles showed that the SM is present in nuclear domains active in DNA replication, transcription and probably also in different steps of mRNA processing (data not shown). In the other hand the SM, N-SMase and SM-synthase activity have been recently demonstrated in an intranuclear [START_REF] Rossi | Signal transducer and activator of transcription 3 and sphingomyelin metabolism in intranuclear complex during cell proliferation[END_REF]. Moreover N-SMase activity plays various roles in different subnuclear fractions. In fact, during cell proliferation induced by partial hepatectomy, the activation of the enzyme present in nuclear membrane changes the level of SM and cholesterol (CHO) [35] modifying the fluidity and the size of the pores [START_REF] Tomassoni | Changes of nuclear membrane fluidity during rat liver regeneration[END_REF] and consequently regulating the mRNA nucleus-cytoplasm efflux [START_REF] Tomassoni | Changes of nuclear membrane lipid composition affect RNA nucleocytoplasmic transport[END_REF]. In the same experimental conditions, the chromatin N-SMase is involved in the DNA structure modification and in the molecular events that occur during the beginning of the S-phase of the cell cycle [START_REF] Albi | Chromatin neutral spingomyelinase and its role in hepatic regeneration[END_REF]. These results were supported in a study in which the ciprofibrate treatment induced liver cell proliferation and the drug withdrawal stimulated the cells to apoptosis [START_REF] Albi | Chromatin sphingomyelin changes in cell proliferation and/or apoptosis induced by ciprofibrate[END_REF]. The authors have shown that the chromatin N-SMase activity increases during cell proliferation when the SMsynthase activity is depressed whereas the chromatin SM content increases during apoptosis as a consequence of SM-synthase activation [START_REF] Albi | Chromatin sphingomyelin changes in cell proliferation and/or apoptosis induced by ciprofibrate[END_REF]. It has been supposed that a decrease in SM may favour the opening of the helix during DNA synthesis whereas the increase of the SM amount may hinder the DNA duplication. Since these results suggested strongly that modifications of intranuclear lipid metabolism occur in relation to cell life, it was possible to hypothesise the existence of a lipid metabolic machinery that takes place inside the nucleus.

In order to investigate about this hypothesis, the aim of the present research was to study the crosstalk between SM and PC metabolism inside the nucleus either in vitro or in vivo during liver regeneration. were used. They were kept at a normal light-dark period and had free access to pelleted food and water. The animals were laparatomized after anaesthesia with 100 mg/Kg Farmotal (Farmitalia, Italy), the liver was perfused via the portal vein with cold 0.25M sucrose, 3.3mM CaCl 2 , 1.0 mM PMSF solution at pH 7.2 and immediately removed. The animals were killed by cervical dislocation. Liver regeneration was induced by hepatectomy which stimulates liver cells to proliferate. Hepatectomy was performed, after anesthesia, between 8-10 a.m. as previously reported [START_REF] Albi | Involvement of nuclear Phosphatidylinositol-dependent Phospholipases C in cell cycle progression during rat liver[END_REF]. Sham operated animals were used as controls. The animals were killed at different times after operation from 0 to 24 hours. All treatments were made according to the international regulation of National Institutes of Health.

Nuclear membrane and Chromatin purification -Nuclear membrane and chromatin were isolated and purified as previously described [START_REF] Albi | Reverse Sphingomyelin-Synthase in Rat Liver Chromatin[END_REF]. For chromatin extraction, purified hepatocyte nuclei were gently suspended for 5 min in 10 mM Tris containing 0.25 M sucrose 1 mM PMSF, 0.3% Triton X-100 adjusted to pH 7.4 with 0.1N NaOH and centrifuged at 5000 g for 5 min in order to remove the outer nuclear membrane. Subsequent four washings of the preparation with hypotonic solution containing 0.075 M NaCl, 0.024 M EDTA and 1 mM PMSF pH 8.0 resulted in the swelling of the nuclei and destruction of inner nuclear membrane. The chromatin extraction was completed by washing the denuded nuclei in progressively less concentrated Tris solutions (50mM, 10 mM, 2mM and 0.4mM) containing 1mM PMSF, pH 8.0. After each washing, the material was recovered by centrifugation at 8000 g for 10 min and it was re-suspended each time with 1-3 strokes of the pestle at low Transcriptional activity of the chromatin -The chromatin transcription was evaluated according to Gould et al. [START_REF] Gould | The assay of globin gene transcription in reconstituted chromatin[END_REF] with the following modifications. The DNA content was evaluated at 260nm. Purified chromatin, corresponding to 20 g DNA, was incubated with 10mM Tris pH 8.0, 25mM MnCl 2 , 150 mM -mercaptoetanol, 6 mM ATP, 6mM CTP, 6mM GTP, 3 mM mercuriuridine, 20 l 3 H-uridine and 6 l RNA polymerase to a final volume of 250 l. Incubation was performed at 37 °C for different time until four hours and it was stopped by adding cold 10 mM Tris and 10% TCA (final concentration). Suspension was filtered with GF/C Wathman filter and it was washed six time with 8% TCA until no radioactivity was recovered in the filtered solution. The radioactivity was measured in counting vials containing 10 ml of HionicFluor with a liquid scintillation analyzer (1500 TRI-Carb) Packard Instrument Company, Meriden, USA. The filter was dried by phone and treated in the counting vials containing 0.5 ml Filtron-X at 60°C for 30 min. When the filter became cold, 20 l of acetic acid and 10 ml of Econofluor were added. The radioactivity was measured as above reported and it was referred to RNA content [START_REF] Schneider | Determination of nucleic acids by pentose analysis[END_REF]. Chromatin transcriptional activity was also evaluated after 60' and 90' of treatment necessary for the PC-SM metabolism study as described below in "experiment 1" and "experiment 2" (cold molecules were used at the same concentration of those labelled).

Lipid analysis -Lipids were extracted from nuclear membrane and chromatin; SM and PC were separated on thin layer silica gel chromatography (TLC), identified in relation to the standard and scraped into test tubes for inorganic phosphorous determination [START_REF] Albi | Reverse Sphingomyelin-Synthase in Rat Liver Chromatin[END_REF]. Ceramide was separated according to Previati et al. [START_REF] Previati | Low nanogram range quantitation of diglycerides and ceramide by high-performance liquid chromatography[END_REF] on bidimensional TLC by using chloroform/methanol/acetic acid, 190:9:1 (v/v) and diethyl ether/acetic acid, 100:1 (v/v) as first and second mobile phase respectively. The spot, identified in relation to the standard, was scraped and eluted by two washing with chloroform/methanol (2:1). The ceramide was dried and dissolved in 20 µl pyridine; then 40 µl benzoyl chloride were added. The sample was heated 1 h at 60°C and the solution was evaporated under an efficient fume hood. 4 ml methanol were added and the sample was heated again 1 h at 70°C. After methanol Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. evaporation, 1 ml hexane and 1 ml Na 2 CO 3 saturated methanol solution was added. The sample was centrifuged at low speed. The lower phase was washed first with 1 ml methanol containing 0.6 HCl and then with pure methanol. The lower phase was evaporated and the residue was dissolved in a known volume of hexane for the analysis by HPLC with a column Lichrosorb Si 60 from Merck (125 x 4 mm, 5 µm) and, as mobile phase, hexane/2-propanol (98:2, v/v, flow rate: 0.5 ml/min) and it was measured at 240 nm. The DAG was derivatized directly in the lipid extract by using Naproxen as a fluorescent label [START_REF] Rastegar | Sensitive analysis of phospholipid molecular species by high-performance liquid chromatography using fluorescent naproxen derivatives of diacylglycerols[END_REF] and it was purified by TLC using petroleum hexane/diethyl ether (75:25, v/v) as mobile phase. The spot, identified in relation to the standard, was scraped and eluted by two washing with 2 ml of acetonitrile. The analysis by HPLC was performed with a column Lichrospher RP18 from Merck (250 x 4.6 mm, 5 µm) using, as mobile phase, acetonitrile/iso-propanol (90:10, v/v, flow rate of 1.5 ml/min) and the fluorescence was detected (exc: 230 nm, em: 352 nm). The total ceramide and DAG content was estimated by the measurement of the area of all identified peaks.

Phosphatidylcholine-sphingomyelin metabolism

The experimental plan is below reported (Fig. 1)

Experiment 1. Effect of nuclear membrane or chromatin endogenous N-SMase on PC-PLC

and SM-synthase activities -1nmol of [ 14 C]-SM was diluted by adding 49 nmol cold SM to a final radioactivity of 1.08 Ci/mol. The preincubation was performed for 60 min at 37 °C with a reaction mixture containing 0.1 M Tris/HCl pH 7.6 or 8.4 for nuclear membrane or chromatin respectively, 6 mM MgCl 2 , 0.1 % Triton X-100, 0.1 mM 14 C-SM and sample suspension in 10 mM Tris + 1 mM PMSF (pH 7.6 and pH 8.4 for nuclear membrane and chromatin respectively) equivalent to 200 µg protein, to a final volume of 0.2 ml. In the control sample, the preincubation was performed without 14 C-SM. At the end of the preincubation, considered 0' time, 0.1 mM 14 C-SM + 0.3 mM 3 H-PC, 0.1mM ceramide, 80 g/ml Na-taurodeoxycholate and 2 mM CaCl 2 were added to a volume final of 0.3 ml (the concentrations of all substances are indicated as final concentrations). The incubation was prolonged for 7.5, 15, 30 min and it was stopped by adding 3.0 ml chloroform/methanol (1:1, v/v). The organic phase was washed with 0.2 volumes of 0.5% NaCl. The next day, the upper phase was removed, measured and 0.5 ml were diluted in counting vials with 10ml Ecoscint A and 1ml water to evaluate 3 H-PPC for PC-PLC activity and 14 C-PPC for N-SMase activity. The lower phase was used to evaluate the SM-synthase activity. It was dried under nitrogen flow and the lipids were re-suspended with chloroform and separated with thin-layer silica gels (TLC) chromatography by using chloroform/methanol/ammonia (65:25:4, by vol) as solvent. In the sample, cold SM was added to the tubes before chromatography in order to better identify the spot. The SM was identified on the basis of its migration related to SM standard, localised with iodine vapour and scraped into counting vials with 10 ml Ecoscint A and 1 ml water. The radioactivity was measured as above reported for the analysis of transcriptional activity of the chromatin. For DAG and ceramide determination, the samples with 1 mg of protein at 0 and 30 min of incubation were prepared. The content of ceramide was calculated in both control and experimental sample considering the ceramide added to the incubation medium. [START_REF] Albi | Phosphatidylcholine-Dependent Phospholipase C in Rat Liver Chromatin[END_REF] and the N-SMase [START_REF] Albi | Chromatin neutral spingomyelinase and its role in hepatic regeneration[END_REF], SM-synthase [START_REF] Albi | Sphingomyelin-Synthase in Rat Liver Nuclear Membrane and Chromatin[END_REF], PC-PLC [START_REF] Albi | Phosphatidylcholine-Dependent Phospholipase C in Rat Liver Chromatin[END_REF], and RSM-synthase [START_REF] Albi | Reverse Sphingomyelin-Synthase in Rat Liver Chromatin[END_REF] activities were assayed after 0, 7.5, 15 and 30 min.

Statistical analysis -Unless otherwise stated means ± SD are given. The significance of the differences between the data was checked by student test.

Results

Nuclear Membrane and Chromatin lipid fraction

The results of lipid content and enzyme activities obtained in purified nuclear membrane and chromatin are reported in the Table 1. In the nuclear membrane, the PC content is about 12 times SM content whereas the DAG only 2 times ceramide content. In fact, the DAG/ceramide ratio is 1.98. The activities of N-SMase and SM-synthase are similar and their value is 50% of that of PC-PLC. The activity of RSM-synthase is very low. In the chromatin, the value of PC is 4.47 times higher of SM value. The DAG is 1.57 higher of ceramide. The activity of N-SMase, similar to that of RSM-synthase, is 9% of the SMsynthase activity. PC-PLC activity value is similar to that of SM-synthase. The results of the lipid composition and enzyme activities in nuclear membrane and chromatin support previous observations [START_REF] Albi | The role of intranuclear lipids[END_REF]. The comparison of the results indicates a strong difference in PC/SM ratio in the two fractions and a very high SMase activity in nuclear membrane whereas the RSMsynthase activity is higher in chromatin.

Characteristics of purified chromatin

The chromatin isolation procedure used here consists of many treatments that remove first the outer nuclear membrane and then the inner nuclear membrane with a progressive and gentle swelling of the nuclear material that permits a good preservation of the chromatin. The purified chromatin remains transcriptionally active for two hours (Fig. 2). Moreover, it is also active after the treatments made for the PC-SM metabolism crosstalk study either after the preincubation time or after incubation time as above reported in the "experiment 1" and "experiment 2" (Fig. 2).

S t a g e 2 ( a ) P O S T -P R I N T

Effect of endogenous N-SMase on PC-PLC and SM-synthase activities and of endogenous PC-PLC on N-SMase and RSM-synthase activities in nuclear membranes

In nuclear membrane, the values of the lipid metabolism enzyme activities in the control sample are linear in the time. After preincubation (0' time) with 14 C-SM (experiment 1) or 3 H-PC (experiment 2), N-SMase activity is 52 + 11 and whereas PC-PLC activity is 97 + 15 nmol/mg protein. The preincubation with 14 C-SM does not modify the PC-PLC and SMsynthase activity in the time with respect to the control (Fig. 3a); moreover the preincubation with 3 H-PC does not influence the N-SMase and RSM-synthase activity (Fig. 3b). No variations are observed in DAG and ceramide content at 30 min of incubation with respect to the control in the experiment 1 (Fig. 5a) whereas an increase of DAG is shown in the experiment 2 (Fig. 6a)

Effect of endogenous N-SMase on PC-PLC and SM-synthase activities and of endogenous PC-PLC on SMase and RSM-synthase activities in chromatin

In chromatin, the values of the lipid metabolism enzyme activities in the control sample are linear in the time. After preincubation (0' time) with 14 C-SM (experiment 1) or 3 H-PC (experiment 2), N-SMase activity is 1200 + 321 whereas PC-PLC is 9560 + 250 pmol/mg protein. The preincubation with 14 C-SM produces a PC-PLC activity increase of 1.63, 6.64 and 5.56 times after 7.5, 15 and 30 min respectively in comparison with the control (Fig. 4a).

At the same times the SM-synthase activity increases of 2.06, 1.99 and 10.94 times (Fig. 4a).

After 30 min the DAG content increases of 3.81 and ceramide content decreases 4.19 times with respect to the control (Fig. 5b). DAG/ceramide ratio is 1.5 and 3.0 after 0 and 30 min respectively in the control whereas the values are 0.93 and 40.03 in the experimental sample.

Therefore endogenous N-SMase activity induces an enrichment of ceramide pool that is responsible for first PC-PLC and after SM-synthase activation enriching the DAG pool and reducing that of ceramide in the chromatin.

The endogenous PC-PLC activity produces a N-SMase activity increase of 1.75, 1.5 and 1.62 times after 7.5, 15 and 30 min respectively compared to the control (Fig. 4b). RSM-synthase activity has the same value at 7.5 min and increases 3.2 times at 15 min and 2.90 times at 30 min with respect to the control (Fig. 4b). Therefore PC-PLC stimulates first N-SMase and then RSM-synthase activity. After 30 min the DAG content decreases 1.76 times and that of ceramide increases 2.90 times with respect to the control. DAG/ceramide ratio is 1. 6b). Therefore in the chromatin PC-PLC activity determines an enrichment of DAG pool that is responsible for first N-SMase and after RSM-synthase activation enriching the ceramide pool and reducing that of DAG.

Effect of D609 on enzyme activities

To better verify the interaction between PC and SM metabolism, incubation of nuclear membrane and chromatin by D609 was performed. The results show that in nuclear membrane the PC-PLC (Fig. 7a) and SM-synthase (Fig. 7b) activity is inhibited 99% and 35% respectively after 30 min in comparison with the control. No modifications are observed for N-SMase (Fig. 7c) and RSM-synthase (Fig. 7d).

In chromatin, D609 treatment induces a PC-PLC activity reduction of 22%, 48% and 40% after 7.5, 15 and 30 min respectively (Fig. 8a). At the same times an inhibition equal to 59%, 75% and 86% for SM-synthase (Fig. 8b), 1.7%, 10% and 48% for N-SMase (Fig. 8c) and 14%, 13% and 45% for RSM-synthase (Fig. 8d) appears. Therefore D609 acts first on SM-synthase and then on PC-PLC; the reduction of DAG pool could be responsible for N-SMase and RSM-synthase activity inhibition.

Phosphatidylcholine-sphingomyelin metabolism during rat liver regeneration

In order to study if the crosstalk of chromatin PC and SM metabolism obtained in vitro could occur in vivo, the activity of PC-PLC, N-SMase, SM-synthase and RSM-synthase has been evaluated in the regenerating rat liver after partial hepatectomy. The results show that in the nuclear membrane PC-PLC and N-SMase activity increases progressively in time until 24 hours, the SM-synthase forms a peak at 18 hours whereas RSM-synthase does not show any variation (Fig. 9a). In chromatin the activity of PC-PLC increases at 12 hours and it is reduced at 18 hours when N-SMase and RSM-synthase activity increases, whereas the SMsynthase activity increases strongly at 24 hours together to a new increase of PC-PLC activity (Fig. 9b). No significant modifications are reported for sham operated animals.

Discussion

The results highlighted clearly that in nuclear membrane the metabolism of PC is independent from that of SM and vice-versa, whereas in chromatin a crosstalk between the two lipid metabolisms exists. To study these metabolic interactions, each enzyme has been activated by adding the substrate of the reaction since until now do not exist physiologic activators or [START_REF] Watanabe | Increase of nuclear ceramide through caspase-3-dependent regulation of the "sphingomyelin cycle" in Fas-induced apoptosis[END_REF]. The authors have studied the whole nuclei whereas our results are referred to the nuclear membrane and chromatin purified from isolated nuclei which have different physico-chemical properties [START_REF] Albi | Chromatin neutral spingomyelinase and its role in hepatic regeneration[END_REF][START_REF] Albi | Sphingomyelin-Synthase in Rat Liver Nuclear Membrane and Chromatin[END_REF][START_REF] Albi | Phosphatidylcholine-Dependent Phospholipase C in Rat Liver Chromatin[END_REF][START_REF] Albi | Reverse Sphingomyelin-Synthase in Rat Liver Chromatin[END_REF]. In chromatin the mechanism of the enzyme regulation was very fine. In fact the N-SMase activity reduces the SM level with consequent enrichment of ceramide pool that is responsible for the stimulation of PC utilization first by PC-PLC and then by SM-synthase activation. The PC-PLC activity degrades PC freeing PPC and DAG; the SM-synthase activity permits to re-synthesise SM by using ceramide free and PC as source of PPC with consequent release of DAG. In this way an early and a later production of DAG with a reduction of ceramide pool occur with consequent increase of DAG/ceramide ratio. Differently the PC-PLC activity determines an enrichment of DAG pool that is responsible for first N-SMase and then RSM-synthase activation. N-SMase degrades SM freeing PPC and ceramide whereas RSM-synthase re-synthesises PC by using DAG free and PPC derived from SM with release of ceramide. Consequently, as above reported for the DAG, an early and a later ceramide production are obtained. Therefore in the chromatin the SM and PC metabolisms are very correlated. The results are supported by the experiment with D609, a drug known to be an inhibitor of PC-PLC [START_REF] Li | Involvement of phosphatidylcholine-selective phospholipase C in activation of mitogen-activated protein kinase pathways in imidazoline receptor antisera-selected protein[END_REF] and SM-synthase [45] also at nuclear level [START_REF] Albi | Sphingomyelin-Synthase in Rat Liver Nuclear Membrane and Chromatin[END_REF]. This effect is evident in nuclear membrane in which the drug acts at the same time on the two enzymes even if with different percentage. No inhibitor effect on the N-SMase or RSM-synthase activity is shown. In chromatin the D609 inhibitor effect on different enzymes is evident at various times. In fact, it has been demonstrated first an inhibition of SM-synthase and then of PC-PLC activity; the last is responsible for a later N-SMase and RSM-synthase inhibition. Therefore a mechanism of internal regulation of lipid metabolism exists in chromatin probably to maintain an equilibrium either between SM and PC or ceramide and DAG when the cells are in the physiological state. To study the possible role of chromatin SM-PC crosstalk in vivo, it has been stimulated liver cell proliferation by partial hepatectomy. In fact, liver regeneration represents a good model of synchronous proliferation in vivo. The hepatocytes enter in G1 phase and begin to synthesise DNA 18 hrs after hepatectomy and divide after 24hrs. Our results show that during liver regeneration the metabolisms of PC and SM localised in nuclear membrane do not are in relation whereas those present in chromatin present a fine regulation mechanism. In fact, the activity of chromatin PC-PLC increases at the beginning of the S-phase and is reduced during S-phase [START_REF] Li | Involvement of phosphatidylcholine-selective phospholipase C in activation of mitogen-activated protein kinase pathways in imidazoline receptor antisera-selected protein[END_REF] or ceramide [45] dependent PKC isoforms. It has been demonstrated that PKC alpha is stimulated by DAG [START_REF] Albi | Nuclear sphingomyelin-synthase and protein kinase C delta in melanoma cells[END_REF] and inhibited by ceramide [45]. In HL-60 cells, the depletion of PKC alpha from the cytosol abolishes the stimulation vitamin D3-dependent of N-SMase, enzyme inhibited by PKC delta [START_REF] Stahelin | Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Cdelta[END_REF]. In fact the PKC delta depletion with specific antibodies increases significantly the SMase activity [START_REF] Stahelin | Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Cdelta[END_REF]. On the other hand the PKC delta is a ceramide activated kinases [START_REF] Abboushi | Ceramide inhibits IL-2 production by preventing protein kinase C-dependent NF-kappaB activation: possible role in protein kinase Ctheta regulation[END_REF]. It is possible that DAG stimulates SM hydrolysis [START_REF] Shen | Lipid-dependent activation of protein kinase C-alpha by normal alcohols[END_REF] by activating PKC alpha or inhibiting PKC delta. Inside the nuclei isolated from rat liver the PLC activity increases PKC alpha [START_REF] Visnjic | Different roles of protein kinase C alpha and delta isoforms in the regulation of neutral sphingomyelinase activity in HL-60 cells[END_REF] whereas in the nuclei isolated from BL6 murine melanoma cells, that do not express the PKC [START_REF] Gopee | Sphingolipid bases and their phosphatases: transient activation and delayed repression of protein kinase C isoforms and their possible involvement in fumonisin B1 cytotoxicity[END_REF], the overexpression of this PKC isoform inhibits the N-SMase activity and stimulates the SM-synthase [START_REF] Schneider | Determination of nucleic acids by pentose analysis[END_REF]. Therefore it is possible to hypothesise that at the nuclear level the ceramide could have inhibitor effect on DAG activated kinases, such as PKC alpha which, for regulatory mechanism, could stimulate PC-PLC increasing the DAG pool. Vice-versa, the DAG could have inhibitor effect on ceramide activated kinases, such as delta isoform with consequent stimulation of N-SMase increasing the ceramide pool. Probably D609 by inhibiting SM-synthase and PC-PLC activity and consequently reducing the level of DAG, does not permit the inhibition of the PKC delta and therefore the N-SMase activity results inhibited.

In conclusion our results show that SM and PC can be synthesised and hydrolysed directly in chromatin with a regulatory mechanism, suggesting that they could have a specific role in relation to cell function. Experimental plan. In the "experiment 1" the preincubation with 14 C-SM induces activation of N-SMase. In the control sample the preincubation was performed without 14 C-SM. The effects of N-SMase activity in the presence of specific substrate on PC-PLC and SM-synthase activity were evaluated at different times of incubation as reported in the "Materials and Methods" section. In the "experiment 2" the preincubation with 3 H-PC induces activation of PC-PLC. In the control sample the preincubation was performed without 3 H-PC. The effects of PC-PLC activity in the presence of specific substrate on N-SMase and RSM-synthase activity were evaluated at different time of incubation as reported in the "Materials and Methods" section.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Nov 2007 as manuscript BJ20070758 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070758 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society complex containing STAT3 and a new-synthesised double-strand RNA, supporting their association with the active chromatin

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Nov 2007 as manuscript BJ20070758 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070758 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society Experimental Procedures Materials -The radioactive SM (choline-methyl 14 C, 54.5 Ci/mol) and PC (L-3-phosphatidyl N-methyl-3 H choline 1,2 dipalmitoyl, 81.0 Ci/mmol) and 3 H-UTP were from Amersham Pharmacia Biotech (Rainham, Essex, UK); GF/C filters were from Whatman (Maidstone, Kent, UK). HionicFluor and Econofluor were from PerkinElmer (Waltham, Massachusetts, USA) and Ecoscint A and Filtron-X were from National Diagnostic (Atlanta, Georgia, U.S.A.). PC, SM, ceramides, diacylglycerol and O-Tricyclodec-9-yl ditiocarbonate potassium salt (D609), phenylmethylsulfonylfluoride (PMSF), ethylendiaminetetracetic acid (EDTA) and RNA polymerase were from Sigma Chemical Co. (St. Louis, Missouri, U.S.A.) Animals -Thirty day old Sprague Dawley rats of either sex (Harlan Nossan, Milano, Italy)

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Nov 2007 as manuscript BJ20070758 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070758 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society velocity. Two washings for each solution were made. Finally the material was re-suspended in 1mM PMSF (a protease inhibitor) brought to pH 8 with ammonium hydroxide and stirred overnight at 4 °C. The solution was then centrifuged at 90000 g for 30 min. The chromatin containing sediment was re-suspended in 10 mM Tris + 1 mM PMSF, pH 8.6.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Nov 2007 as manuscript BJ20070758 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070758

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Nov 2007 as manuscript BJ20070758 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070758 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Experiment 2 .

 2 Effect of nuclear membrane or chromatin PC-PLC endogenous activity on N-SMase and RSM-synthase -1nmol of [ 3 H]-PC was diluted by adding 99 nmol cold PC to a final radioactivity of 1.27 Ci/mol. The preincubation was performed for 60 min at 37 °C with a reaction mixture containing 0.1 M Tris/HCl pH 7.6 or 8.4 for nuclear membrane or chromatin respectively, 0.3 mM 3 H-PC, 2 mM CaCl 2 , 0.1% Triton X-100, sample suspension in 10 mM Tris + 1 mM PMSF (pH 7.6 and pH 8.4 for nuclear membrane and chromatin respectively) equivalent to 200 g protein, to a final volume of 0.2 ml. In the control sample, incubation was performed without 3 H-PC. At the end of the preincubation, considered 0' time, 0.3 mM 3 H-PC + 0.3 mM 14 C-SM, 0.3 mM DAG, and 6 mM MgCl 2 were added to a volume final of 0.3 ml. (the concentrations of all substances are indicated as final concentrations). The incubation was prolonged for 7.5, 15, 30 min and it was stopped by adding 3.0 ml chloroform/methanol (1:1, v/v). The organic phase was washed with 0.2 volumes of 0.5% NaCl. The next day, the upper phase was removed, measured and 0.5 ml were diluted in counting vials with 10ml Ecoscint A and 1ml water to evaluate 3 H-PPC for PC-PLC and 14 C-PPC for SMase. The lower phase was used for RSM-synthase evaluation by using the same method of that used for SM-synthase evaluation but utilizing cold PC to identify the spot. The radioactivity was measured and DAG and ceramide content was evaluated as above reported. The content of DAG was calculated in both control and experimental sample considering the DAG added to the incubation medium S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Nov 2007 as manuscript BJ20070758 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070758 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society D609 treatment. The nuclear membrane or chromatin corresponding to 200 g protein were incubated with 200 nmol of tricyclodecan-9-yl-xanthogenate (D609)

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64 and 3 .

 3 01 after 0 and 30 min in the control whereas in the experimental sample the values are 4.36 S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Nov 2007 as manuscript BJ20070758 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070758 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society and 0.59 respectively (Fig.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Nov 2007 as manuscript BJ20070758 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070758 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society inducers. Watanabe et al. have shown that Fas activates the N-SMase and inhibits SMsynthase in the nuclei isolated from human leukaemia Jurkat T-lymphoid leukaemia cells

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Nov 2007 as manuscript BJ20070758 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070758 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society when N-SMase and RSM-synthase activities increase whereas the SM-synthase increases strongly only at the end of the S-phase. At the moment we do not have sufficient data to describe the exact mechanism of action for ceramide derived from N-SMase on PC-PLC and for DAG derived from PC-PLC on N-SMase. It is possible to suppose the involvement of different DAG

Fig. 1

 1 Fig.1

Fig. 2

 2 Fig. 2 Chromatin transcription. Incubation was performed as reported in the "Materials and Methods" section. The values are expressed as cpm/mg RNA and the values represent the mean + S.D. of four separated experiments performed in duplicate. ** P< 0.01 versus 0 time. Control= chromatin incubated without SM or PC; SM= chromatin incubated with 0.1 mM SM; PC= chromatin incubated with 0.3 mM PC; Exp. 1= experiment 1 and Exp. 2= experiment 2 are reported in the "Materials and Methods" section.

Fig. 3 SphingomyelinFig. 4 SphingomyelinFig. 5 Diacylglycerol

 345 Fig. 3 Sphingomyelin and Phosphatidylcholine metabolism enzymes in nuclear membrane at different incubation times: a) experiment 1: effect of endogenous sphingomyelinase activity in the presence of specific substrate; b) experiment 2: effect of endogenous phosphatidylcholine-dependent phospholipase C activity in the presence of specific substrate. The enzyme activities are expressed as nmol/mg protein and the values represent the mean + S.D. of five separated experiments performed in duplicate. In (b) the right ordinate is referred to RSM-synthase activity (pmol/mg protein). PC-PLC: phosphatidylcholine-dependent S t a g e 2 ( a ) P O S T -P R I N T

Fig. 6 DiacylglycerolFig. 7 S

 67 Fig. 6 Diacylglycerol and ceramide content in a) nuclear membrane and b) chromatin after endogenous phosphatidylcholine-specific phospholipase C activity in the presence of specific substrate. The data represent the values obtained after 30 min of incubation and were calculated taking in account the diacylglycerol added. The values were expressed as nmol/mg protein and represent the mean + S.D. of five separated experiments performed in duplicate. Significance ** P< 0.01 versus control. DAG, diacylglycerol.

Fig. 8 Effect

 8 Fig. 8 Effect of the D609 treatment on lipid enzymatic activities present in Chromatin. a) PC-PLC, phosphatidylcholine-dependent phospholipase C; b) SM-synthase, sphingomyelin-synthase; c) N-SMase, neutral-sphingomyelinase; d) RSM-synthase, reverse sphingomyelin-synthase. C= control sample incubated without D609; Ex= experimental sample incubated in the presence of D609. The enzyme activities were expressed as pmol /mg protein and the values represent the mean + S.D. of four separated experiments performed in duplicate. Significance ** P< 0.01 versus control.

Fig. 9 Behaviour

 9 Fig.9 Behaviour of lipid enzyme activities during rat liver regeneration. Cell proliferation is induced by partial hepatectomy as reported in the "Materials and Methods"; a) nuclear membrane and b) chromatin were prepared at different times after operation. The enzyme activities were expressed as pmol /mg protein/min and the values represent the mean + S.D. of four separated experiments performed in duplicate. In (a) the right ordinate is referred to PC-PLC activity. Significance ** P< 0.01 versus 0 time. PC-PLC: phosphatidylcholinedependent phospholipase C; SM-synthase, sphingomyelin-synthase; N-SMase, neutralsphingomyelinase; RSM-synthase, reverse sphingomyelin-synthase

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Acknowledgements

Support of this work by Ministero dell' Università e Ricerca, Agenzia Spaziale Italiana (ASI) , and Fondazione Cassa di Risparmio di Perugia is gratefully acknowledged. We thank Bartoccini E., Bernardini I., Cascianelli G., Marini F. for the technical assistance S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Nov 2007 as manuscript BJ20070758 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070758 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society