BBA - General Subjects (v.1770, #1)

BBA in the year 2007 by Dennis E. Vance (3-4).

Transition of ovalbumin to thermostable structure entails conformational changes involving the reactive center loop by Hiroshi Shinohara; Masahisa Horiuchi; Mamoru Sato; Junichi Kurisaki; Takahiro Kusakabe; Katsumi Koga; Yuji Minami; Takayoshi Aoki; Ikunosin Kato; Yasushi Sugimoto (5-11).
Ovalbumin is a serpin without inhibitory activity against proteases. During embryonic development, ovalbumin in the native (N) form undergoes changes and takes a heat-stable form, which was previously named HS-ovalbumin. It has been known that N-ovalbumin is artificially converted to another thermostable form called S-ovalbumin by heating at an alkaline pH. Here, we characterized further the three ovalbumin forms, N, HS, and S. The epitope of the monoclonal antibody 2B3/2H11, which recognizes N- and HS-ovalbumin but not S-ovalbumin, was found to reside in the region Glu–Val–Val–Gly–Ala–Ser–Glu–Ala–Gly–Val–Asp–Ala–Ala–Ser–Val–Ser–Glu–Glu–Phe–Arg, which corresponds to 340–359 of amino acid residues and is contained in the reactive center loop (RCL). Removal of RCL by elastase or subtilisin mitigated binding of the antibody. Dephosphorylation experiments indicated that the phosphorylated Ser-344 residue located on RCL is crucial for the epitope recognition. We suggest that the shift to the heat-stable form of ovalbumin accompanies a movement of RCL.
Keywords: Ovalbumin; Thermostability; Reactive center loop; Serpin; Gallus gallus;

Electroporation is a process where increased permeability of cells exposed to an electric field is observed. It is used in many biomedical applications including electrogene transfection and electrochemotherapy. Although the increased permeability of the membrane is believed to be the result of pores due to an induced transmembrane voltage U m, the exact molecular mechanisms are not fully explained.In this study we analyze transient conductivity changes during the electric pulses and increased membrane permeability for ions and molecules after the pulses in order to determine which parameters affect stabilization of pores, and to analyze the relation between transient pores and long-lived transport pores. By quantifying ion diffusion, fraction of transport pores f per was obtained. A simple model, which assumes a quadratic dependence of f per on E in the area where U m  >  U c very accurately describes experimental values, suggesting that f per increases with higher electric field due to larger permeabilized area and due to higher energy available for pore formation. The fraction of transport pores increases also with the number of pulses N, which suggest that each pulse contributes to formation of more and/or larger stable transport pores, whereas the number of transient pores does not depend on N.
Keywords: Electroporation; Ion diffusion; Conductivity; Cell suspension; Electrochemotherapy;

Characterization of α-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity by Rakesh Mohan Kestwal; Emadeldin Hassan E. Konozy; Chwan-Deng Hsiao; Maria Cristina Roque-Barreira; Shobhana V. Bhide (24-28).
α-mannosidase from Erythrina indica seeds is a Zn2+ dependent glycoprotein with 8.6% carbohydrate. The enzyme has a temperature optimum of 50 °C and energy of activation calculated from Arrhenius plot was found to be 23 kJ mol− 1. N-terminal sequence up to five amino acid residues was found to be DTQEN (Asp, Thr, Gln, Glu, and Asn). In chemical modification studies treatment of the enzyme with NBS led to total loss of enzyme activity and modification of a single tryptophan residue led to inactivation. Fluorescence studies over a pH range of 3–8 have shown tryptophan residue to be in highly hydrophobic environment and pH change did not bring about any appreciable change in its environment. Far-UV CD spectrum indicated predominance of α-helical structure in the enzyme. α-Mannosidase from E indica exhibits immunological identity with α-mannosidase from Canavalia ensiformis but not with the same enzyme from Glycine max and Cicer arietinum. Incubation of E. indica seed lectin with α-mannosidase resulted in 35% increase in its activity, while no such activation was observed for acid phosphatase from E. indica. Lectin induced activation of α-mannosidase could be completely abolished in presence of lactose, a sugar specific for lectin.
Keywords: Erythrina indica; α-mannosidase; Lectin; Leguminosae; Characterization; Protein body; Interactions; Activation;

Specific peptide patterns of follicular fluids at different growth stages analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry by Ai-Xia Liu; Yi-Min Zhu; Qiong Luo; Yan-Ting Wu; Hui-Juan Gao; Xiao-Ming Zhu; Chen-Ming Xu; He-Feng Huang (29-38).
Human follicular fluid (HFF) has been suggested to influence oocyte development potential, and some of HFF proteins may be potential markers for oocyte maturation during follicular development. Using matrix-assisted laser desorption /ionization-time of flight mass spectrometry (MALDI-TOF MS), the presence of specific peptide peaks in HFF which could represent the follicle development potential was evaluated. HFF from different developmental stages were first digested and the resultant peptide mixtures were directly analyzed by MALDI-TOF MS. It was shown that the frequencies of specific peaks demonstrated higher reproducibility than peak intensities after multiple measurements (≥ 6 times) per sample. Using this approach, a reliable peak list for each different sample could be generated by combining the information from multiple measurements. By comparing the peak lists from different samples at different growth stages, we found that 5 specific peaks appeared in the 100% frequency category of 6 replicates in all the HFF samples containing mature oocyte. Similarly, such 25 peptide peaks were also identified for HFF containing immature oocyte. These specific peaks could be used to distinguish HFF from different stages as biomarkers related to follicle development and maturation. After searching the protein database, some proteins that are known to be involved in the development and maturation of oocyte were identified, such as apolipoprotein A-I, collagen type IV, integrin, et al. Identification of such proteins in our experiment further proved that the direct analysis of tryptic digests could be of practical value.
Keywords: Follicular fluid; Matrix-assisted laser desorption/ionization-time of flight; Peptide; Biomarker;

Short chain fatty acids including butyrate exhibit wide variety of biological effects towards cell growth, morphology and gene expression. In this report, we study the mechanism by which butyrate (BuA) modulates the expression of protein phosphatase when treated to the cells. As a model system, we used Ehrlich Ascites Tumor (EAT) cells in which BuA-treatment induces expression of a protein phosphatase enzyme. Subsequently, BuA-induced protein phosphatase has been biochemically purified and characterized. Further, pretreatment of caspase-3 inhibitor abolished the activity of BuA-induced protein phosphatase indicating the involvement of caspase-3 in the activation of BuA-induced protein phosphatase. In addition, the relationship between BuA-induced protein phosphatase and apoptosis has been verified. Activation of endonuclease-II has been shown in BuA-treated EAT cells and that activity was completely inhibited by sodium orthovanadate, a tyrosine phosphatase inhibitor suggesting that endonuclease-II may serve as a possible down-stream target for BuA-induced protein phosphatase. Together, the data suggest that activation of protein phosphatase may be an early and essential step in BuA-mediated apoptotic signaling pathway in EAT cells.
Keywords: Butyrate; Protein phosphatase; Caspase-3; Endonuclease-II; Apoptosis; EAT cell;

The endocrine regulation of milk lipid synthesis and secretion in tammar wallaby (Macropus eugenii) by Joly H.L. Kwek; Chakra Wijesundera; Matthew R. Digby; Kevin R. Nicholas (48-54).
Lipids in tammar milk are predominantly triacylglycerols, and the fatty acid composition varies during the lactation cycle. Little is known about the regulation of their synthesis. This study investigates the endocrine regulation of lipid synthesis in mammary explants from pregnant tammars. Treatment of mammary explants with insulin resulted in a high level of lipid synthesis, but the lipids accumulated in the cytosol. Culture with prolactin resulted in a small increase in lipid synthesis, but electron microscopy showed lipid globules were synthesized in the mammary epithelial cells and secreted into the lumen. Culture with both insulin and prolactin demonstrated elevated levels of synthesis and secretion of lipid. Analysis of the type of fatty acids synthesized in these mammary explants showed that the initiation of synthesis of C16:0, which also occurs in the first week of lactation, could be reproduced in the pregnant explants cultured with prolactin alone. However, treatment of mammary explants with hydrocortisone did not show a significant effect on lipid synthesis, secretion or the fatty acid synthesized. These results provide new information identifying the role of insulin and prolactin in regulating milk lipid synthesis and secretion in the tammar.
Keywords: Tammar wallaby; Mammary gland; Lipid; Fatty acid; Synthesis; Secretion;

A novel thermostable α-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: Purification and characterization by Judit M. Rezessy-Szabó; Quang D. Nguyen; Ágoston Hoschke; Christophe Braet; Gyöngyi Hajós; Marc Claeyssens (55-62).
High levels of an extracellular α-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH4)2SO4 fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric α-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5–5.5 and 65 °C, respectively. The purified enzyme retains more than 90% of its activity at 45 °C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-α-galactopyranoside, raffinose and stachyose and very similar K m values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn++ ions activates enzyme activity, whereas inhibitory effects can be observed with Ca++, Zn++ and Hg++. Five min incubation at 65° with 10 mM Ag+ results in complete inactivation of the purified α-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the α-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36.
Keywords: α-Galactosidase; Purification; Thermophilic fungus; Thermomyces lanuginosus;

PKC inhibition is involved in trichosanthin-induced apoptosis in human chronic myeloid leukemia cell line K562 by Jie Li; Xuechun Xia; Huiling Nie; Mark A. Smith; Xiongwei Zhu (63-70).
Trichosanthin (TCS), a type I ribosome-inactivating protein, induces cell death in various cell types including several tumor cell lines. However, the mechanism remains largely uncharacterized. In this study, we investigated the possible mechanism underlying its cytotoxicity by using human chronic myeloid leukemia cell line K562. We found that TCS induced apoptosis in K562 cells in a time- and concentration-dependent manner and can be blocked by caspase-3 inhibitors. Interestingly, TCS treatment induced a transient elevation in intracellular calcium concentration and a slow increase in reactive oxygen species production, while calcium chelators and antioxidants had no obvious effect on TCS-induced apoptosis, suggesting that calcium changes and reactive oxygen species may not be involved in TCS-mediated apoptosis in K562 cells. Instead we found that TCS partly inhibited PKC activity. Indeed, the PKC activator, PMA, inhibited while the PKC inhibitor, calphostin c, enhanced TCS-induced apoptosis. These PKC modulators had similar effects on TCS-induced cleavage of caspase-3, and caspase-3 inhibitors prevented calphostin c-enhanced apoptosis induced by TCS. In summary, we conclude that TCS induces apoptosis in K562 cells partly via PKC inhibition and caspase-3 activation.
Keywords: Trichosanthin; Apoptosis; Calcium; PKC; Caspase-3;

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that plays an important role in the regulation of cell proliferation and protein synthesis through the activation of its downstream target ribosomal p70 S6 kinase (p70S6K). The levels of p-mTOR are regulated by the protein kinase B (Akt/PKB). Therefore, the effects of insulin and rapamycin (an inhibitor of mTOR) on the phosphorylation of mTOR (Ser 2448) and p70S6K (Thr 389) as well as on cell proliferation in parental HepG2 cells and HepG2 cells overexpressing constitutively active Akt/PKB (HepG2-CA-Akt/PKB) were studied. Insulin increased the levels of phosphorylated mTOR and p70S6K in both the cell lines. Rapamycin treatment partially decreased the phosphorylation of mTOR but completely abolished the phosphorylation of p70S6K in the absence as well as presence of insulin in both cell lines. The effect of insulin and rapamycin on the cell proliferation in both cell lines was further studied. In the presence of serum, parental HepG2 cells and HepG2-CA-Akt/PKB showed an increase in cell proliferation until 120 and 168 h respectively. Rapamycin inhibited cell proliferation under all experimental conditions more evident under serum deprived conditions. Parental HepG2 cells showed decline in the cell proliferation after 48 h and the presence of insulin prolonged cell survival until 120 h and this effect were also inhibited by rapamycin under serum deprived conditions. On the contrary, HepG2-CA-Akt/PKB cells continued proliferation until 192 h. The effects of insulin on cell proliferation were more pronounced in parental HepG2 cells as compared to HepG2-CA-Akt/PKB cells. Long term effects of rapamcyin significantly decreased the levels of p-mTOR (Ser 2448) both in the presence and absence of insulin in these cells. A positive correlation between the levels of p-mTOR (Ser2448) and cell proliferation was observed (99% confidence interval, r 2  = 0.525, p  < 0.0001). These results suggest that rapamycin causes a decline in the cell growth through the inhibition of mTOR.
Keywords: Human liver carcinoma cells; Constitutively active Akt/PKB; mTOR; p70S6k; Rapamycin;

Mass spectrometry identification of circulating alpha-1-B glycoprotein, increased in aged female C57BL/6 mice by John R. Stehle; Mark E. Weeks; Kai Lin; Mark C. Willingham; Amy M. Hicks; John F. Timms; Zheng Cui (79-86).
In this study, we surveyed the profiles of mouse circulating proteins by 2-dimensional SDS-PAGE in different strains, sexes and ages. Among visible protein spots on 2-D gels with silver-staining, we identified a unique set of 7 seemingly-related proteins whose levels were consistently elevated in older C57BL/6 female mice. This set of 7 proteins was absent in C57BL/6 males or in BALB/c mice of either sex of any age. When C57BL/6 female mice were crossed with BALB/c males, the age-related increase of these proteins became sporadic and not linear in the F1 offspring. All 7 spots of this protein group were picked and subjected to identification by mass spectrometric analysis after tryptic digestion. The results showed that all 7 spots were different isoforms of α1B-glycoprotein with different degrees of post-translational modifications, such as phosphorylation. These results suggest that α1B-glycoprotein changes in mice in a sex and age dependent manner.
Keywords: Alpha-1-beta glycoprotein; Mouse; Aging; 2D SDS-PAGE; Plasma proteins; Peritoneal lavage;

Purification of properoxinectin, a myeloperoxidase homologue and its activation to a cell adhesion molecule by Xionghui Lin; Lage Cerenius; Bok Luel Lee; Kenneth Söderhäll (87-93).
Peroxidases are important mediators of innate immune reactions throughout the animal kingdom. In many arthropods a myeloperoxidase homologue, peroxinectin, is known to function as a cell adhesion factor and an opsonin. Here, we report in the freshwater crayfish Pacifastacus leniusculus the isolation of properoxinectin, inactive in cell adhesion, and we also show that properoxinectin is produced in the mature blood cells whereas the hematopoietic tissue contains very little of this protein. Both properoxinectin and peroxinectin are catalytically active as peroxidases, at least when using low molecular weight substrates. The extracellular processing of properoxinectin into an active cell adhesion protein was found to involve proteolytic steps shared with the prophenoloxidase activating system to yield catalytically active phenoloxidase. Thus, the regulation of activities by two ancient metalloproteins, both potentially producing highly toxic substances aimed at pathogens, is carried out by limited proteolysis. The proteolytic processing is triggered in the presence of microbial compounds such as beta-glucans or lipopolysaccharide after the release of properoxinectin and prophenoloxidase activating serine proteinases from the blood cells.
Keywords: Peroxidase; Prophenoloxidase; Serine proteinase; Hemocyte;

It is known that subtilisin shows poor transesterification activity in ionic liquids (ILs). The present work, taking subtilisin as the system, explores approaches for biocatalyst preparations, which are capable of yielding higher/adequate transesterification activity in these solvents. Of all the approaches tried, enzyme precipitated and rinsed with n-propanol (EPRP) gave the best results (about 10,000 times increase in initial rates in 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]) over what is obtained with pH tuned lyophilized powders). In case of water soluble ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF4]), pH tuned lyophilized subtilisin did not show any transesterification activity. EPRP, however, gave an initial rate (for transesterification) of 2.78 mmol mg− 1 h− 1.
Keywords: Cryoprotectant; Ionic liquids; Lyoprotectant; Propanol rinsed enzyme preparation; Subtilisin; Three phase partitioning;

The adaptation to extreme concentrations of Ca2+ and its consequence on the properties of the 45Ca2+ transport were studied in submerged mycelia of Trichoderma viride. The adaptation to low [Ca2+]o did not cause changes in kinetic parameters of the 45Ca2+ influx but the adaptation to high [Ca2+]o increased the K M(Ca2+). The V max of the 45Ca2+ influx decreased with the age of (non-adapted) mycelia with concomitant decrease of the K M(Ca2+) these changes were prevented in mycelia adapted to high Ca2+. High [Ca2+]o decreased the stimulation by the uncoupler, 3, 3′, 4′, 5-tetrachloro salicylanilide (TCS) (30 μM), as compared to the control, whereas the Ca2+ chelator, EGTA, stimulated it. In the aged mycelia, the stimulation by TCS of the 45Ca2+ influx faded away, in parallel with the activity of the H+-ATPase. The 45Ca2+ efflux from mycelia was affected by TCS in a similar way as the 45Ca2+ influx. The results demonstrate the adaptive responses of transport processes participating in the mycelial Ca2+ homeostasis and ageing are in agreement with a notion that both Ca2+-influx and-efflux are coupled by the H+-homeostasis at the plasma membrane.
Keywords: Trichoderma viride; Ageing; 45Ca2+ influx; Adaptation; Proton-motive force; Uncoupler;

A lectin from the hemolymph of the banana shrimp Fenneropenaeus merguiensis was purified by affinity chromatography on a fetuin-agarose column following by gel filtration on a Superose-12 column. The native molecular mass of purified F. merguiensis lectin (FmL) determined by gel filtration was 316.2 kDa and its carbohydrate content was estimated to be 4.4%. By SDS-PAGE analysis, purified FmL consisted of 32.3 kDa and 30.9 kDa subunits. These data suggest that this lectin is an oligomer. Two-dimensional electrophoresis showed that it had a pI value of 6.0 and was mainly composed of glycine, serine, histidine, glutamic acids and glutamine, with relatively lower amounts of methionine and tyrosine. Purified FmL expressed higher agglutination activity against rabbit and rat erythrocytes than with those from human, and its activity was Ca2+-dependent. The hemagglutinating activity of FmL was stable up to 55 °C and at pH 7.5–8. N-acetylated sugars, such as ManNAc, GlcNAc, GalNAc, and NeuNAc were strong inhibitors of the FmL induced hemagglutinating activity with NeuNAc being most effective. Porcine stomach mucin and fetuin were the most potent inhibitors of FmL. Purified FmL caused selective agglutination of Vibrio harveyi, and Vibrio parahemolyticus both pathogens of this Penaeus species and to a lesser extent Vibrio vulnificus but had no effect on the non-pathogenic strains; Vibrio cholerae, Salmonella typhi and Escherichia coli. Its bacterial agglutination was also completely inhibited by NeuNAc, mucin, fetuin and also anti-FmL antibody. This observation indicates that FmL may contribute to the defense response of this species of penaeid shrimps to potentially pathogenic bacteria.
Keywords: Lectin; Crustacean; Fenneropenaeus merguiensis; Hemolymph;

The regulation of β-1,3 galactosyltransferase (3βGalT) and β-1,4 galactosyltransferase enzymatic (4βGalT) activities in the mammary gland of the tammar wallaby (Macropus eugenii) have been characterised. These two β-galactosyltransferases are active at different times during the lactation cycle and play a central role in regulating the carbohydrate composition in tammar milk, which changes progressively throughout lactation to assist the physiological development of the altrical young. The 4βGalT activity was present at parturition and increased 3-fold by day 10 of lactation (d10L), whereas 3βGalT activity was barely detectable at day d5L and then increased 6-fold by d10L. This increase in activity of both enzymes was sucking dependent. While 3βGalT activity was not observed in the mammary gland prior to d7L, this activity was found in mammary explants from late pregnant tammar cultured with insulin, hydrocortisone and prolactin (IFP) and was further stimulated by the addition of tri-iodothyronine (T) and 17β-oestradiol (E). The activity of 4βGalT in these explants was stimulated maximally with IFP. These data suggest the temporal activity of both 3βGalT and 4βGalT is most likely regulated by both endocrine stimuli and factors intrinsic to the mammary gland.
Keywords: β-1,3 Galactosyltransferase; β-1,4 Galactosyltransferase; Oligosaccharide; Lactation; Mammary gland; Regulation;

We demonstrated earlier that the gene HAS1 is inactive in resting type-B-synoviocytes but can be readily activated by a series of proinflammatory cytokines including IL-1β. Here we show that in type-B-synoviocytes mRNA levels for the gene COX-2 increase more than 200-fold in response to IL-1β treatment, whereas COX-1 mRNA levels remain virtually unchanged. We tested a series of eicosanoids and demonstrate that PGE2 is a very potent activator of HAS1 in synoviocytes. While μmol concentrations of PGI2 are required to activate HAS1, low nmol concentrations of PGE2 are sufficient. In addition, while two thromboxane A2 analogs moderately activated HAS1 at higher concentrations, the lipoxygenase pathway product LTB4 was without effect. A series of COX inhibitors blocked IL-1β induced HAS1 activation. Similarly, sodium salicylate (NaSal) also suppressed IL-1β induced HAS1 activation. Furthermore, electrophoretic mobility shift assays and PGE2 ELISA experiments demonstrate that NaSal completely prevents PGE2 release but does not interfere with NF-κB translocation. PGE2 is a very powerful activator of HAS1 transcription and translation. Such data indicate that the effect of IL-1β on HAS1 is mediated by prostaglandins. Additionally, NaSal is a potent suppressor of HAS1 activation. These findings point towards HAS1 as a gene of importance in inflammation.
Keywords: Hyaluronan; Hyaluronan synthases; Inflammation; Rheumatoid arthritis; Synoviocytes; Prostaglandins;

Bovine glutathione transferase A1-1 (bGST A1-1) and human GST A3-3 (hGST A3-3) share both high amino acid sequence similarity and selective expression in steroidogenic organs. hGST A3-3 is the most efficient steroid isomerase known in mammals, and is thought to catalyze isomerization reactions in the biosynthesis of steroid hormones. We observed that four out of five residues essential to the high steroid isomerase activity of hGST A3-3 are conserved in bGST A1-1. The bovine GST was cloned, heterologously expressed, and purified to homogeneity. Its specific activity towards classical GST substrates and two steroids, Δ5-androstene-3,17-dione and Δ5-pregnene-3,20-dione, was studied, and the steady-state kinetic parameters with the steroids were determined. We find that bGST A1-1 exhibits enzymatic activities comparable to those of hGST A3-3 towards non-steroid substrates. However, the bovine enzyme had 100 times lower catalytic efficiency in steroid isomerization reactions than the human GST. Nevertheless, bGST A1-1 was found as efficient as bovine 3β-hydroxysteroid dehydrogenase as a steroid isomerase. We discuss likely reasons for the contrasting steroid isomerase activities of bGST A1-1 and hGST A3-3, and alternative roles of bGST A1-1.
Keywords: Alpha-class glutathione transferases; Bovine GST A1-1; Steroid double-bond isomerase; Steroidogenesis; Steroidogenic organ;

New anti angiogenesis developments through electro-immunization: Optimization by in vivo optical imaging of intradermal electrogenetransfer by Sandrine Pedron-Mazoyer; Jean Plouët; Laetitia Hellaudais; Justin Teissie; Muriel Golzio (137-142).
Direct application of high voltage electric pulses of milliseconds duration to the skin of a mouse enhances in vivo intradermal delivery of injected therapeutic molecules such as DNA. The efficacy of gene transfer and expression is dependent on electrical parameters. DNA electrotransfer in tissues increases the associated DNA expression vaccine potency. This protocol is called “electro-immunization”. In the present study, we report a new strategy for optimizing electro-immunization. In vivo fluorescence imaging was used to detect the expression of a fluorescent protein (DsRed) and therefore allowed rapid optimization of the protocol. In vivo electrogenetransfer in the skin was well tolerated and DsRed expression was followed for over 2 weeks. Expression was voltage dependent under our conditions. Parameters were selected giving the highest level of expression. Under these optimized conditions, electrotransfer of a plasmid encoding VEGF was evaluated for its immune response as a gene therapy of interest involved in anti-angiogenic strategies. Anti VEGF 165 antibodies in sera of mice were evaluated by ELISA and compared to those obtained after conventional immunization. Comparable titres of antibodies were obtained in both groups. An IgG2a predominance was found in mice immunized with the plasmid whereas a IgG1 predominance was observed in mice immunized classically. Skin electro-immunization is therefore shown as a good route for DNA immunization for anti-angiogenesis concern.
Keywords: Electroporation; Angiogenesis; DNA vaccine; VEGF; DsRed; In vivo imaging;

Involvement of cytoplasmic membrane damage in the copper (II)-dependent cytotoxicity of a novel naturally occurring tripyrrole by Mahesh Subramanian; Ramesh Chander; Malini Krishna; Subrata Chattopadhyay (143-149).
In the presence of a nonlethal concentration of Cu(II), washed Escherichia coli ATCC8739 cells were killed by a novel tripyrrole 1, isolated as a red pigment from the Serratia sp. Cell killing was accompanied by a depletion in the potassium pools of the cells due to the damage to the cytoplasmic membrane, without any detectable DNA damage as revealed by the transformed plasmid DNA and phage induction assay. This revealed that the bactericidal activity of compound 1 in the presence of Cu(II) results from membrane damage. Induction of endogenous catalase in the E. coli cells increased their resistance against the combination of compound 1 and Cu(II). Although compound 1 alone generated large amount of reactive oxygen species (ROS), it did not show any cell killing against E. coli in the absence of Cu(II). The Cu(II)-dependent bactericidal activity of compound 1 was suppressed by ethylenediaminetetraacetate, bathocuproine, catalase and superoxide disumutase (SOD), but not by dimethyl sulfoxide. These findings suggest that recycling redox reactions between Cu(II) and Cu(I), involving compound 1 and hydrogen peroxide on the cell surface, must be important in the mechanism of the killing. Compound 1 alone showed selective bactericidal activity against the gram positive bacterium, Bacillus cereus ATCC 6630, possibly due to its differential cellular transport.
Keywords: Copper (II); Escherichia coli; Bactericidal activity; Potassium; Catalase; SOD; Free radical; Tripyrrole;

Isolation and characterization of a fish F-type lectin from gilt head bream (Sparus aurata) serum by Matteo Cammarata; Gigliola Benenati; Eric W. Odom; Giuseppina Salerno; Aiti Vizzini; Gerardo R. Vasta; Nicolò Parrinello (150-155).
A novel fucose-binding lectin, designated SauFBP32, was purified by affinity chromatography on fucose–agarose, from the serum of the gilt head bream Sparus aurata. Electrophoretic mobility of the subunit revealed apparent molecular weights of 35 and 30 kDa under reducing and non-reducing conditions, respectively. Size exclusion analysis suggests that the native lectin is a monomer under the selected experimental conditions. Agglutinating activity towards rabbit erythrocytes was not significantly modified by addition of calcium or EDTA; activity was optimal at 37 °C, retained partial activity by treatment at 70 °C, and was fully inactivated at 90 °C. On western blot analysis, SauFBP showed intense cross-reactivity with antibodies specific for a sea bass (Dicentrarchus labrax) fucose-binding lectin. In addition, the similarity of the N-terminal sequence and a partial coding domain to teleost F-type lectins suggests that SauFBP32 is a member of this emerging family of lectins.
Keywords: F-type lectin; Sparus aurata; Dicentrarchus labrax; Teleost; Serum hemagglutinins;

Acknowledgement (156-162).