BBA - General Subjects (v.1770, #2)

Mass spectrometric evidence of covalently-bound tetrahydrolipstatin at the catalytic serine of Streptomyces rimosus lipase by Ivana Leščić Ašler; Martin Zehl; Filip Kovačić; Roland Müller; Marija Abramić; Günter Allmaier; Biserka Kojić-Prodić (163-170).
We have recently detected that the lipase from Streptomyces rimosus belongs to a large but poorly characterised family of SGNH hydrolases having the αβα-fold. Our biochemical characterisation relates to the specific inhibition of an extracellular lipase from Streptomyces rimosus (SRL, 24.2 kDa, Q93MW7) by the preincubation method with tetrahydrolipstatin (THL). In high molar excess (THL/SRL = 590 at 25 °C, pH = 7.0) and after 2 h of incubation in an aqueous system, 56% of the enzyme inhibition was reached. Under the same conditions and in the presence of 50% (v/v) 2-propanol/water, 71% enzyme inhibition was obtained. Kinetic measurements are in agreement with pseudo-first-order kinetics. The nucleophilic attack of the catalytic serine residue 10 of SRL occurs via an opening of the β-lactone ring of tetrahydrolipstatin and formation of a covalent ester bond. The intact covalent complex of SRL-inhibitor was analysed by ESI and vacuum MALDI mass spectrometry and, furthermore, the exact covalent THL linkage was determined by vacuum MALDI high-energy collision-induced dissociation tandem mass spectrometry.
Keywords: Streptomyces rimosus, Extracellular SGNH-lipase; Tetrahydrolipstatin; Covalent inhibition; MALDI tandem mass spectrometry; ESI mass spectrometry; Capillary-gel-electrophoresis-on-the-chip; Kinetics;

Diversity in the degree of sulfation and chain length of the glycosaminoglycan moiety of urinary trypsin inhibitor isomers by Ikuko Kakizaki; Ryouki Takahashi; Nobuyuki Ibori; Kaoru Kojima; Teruno Takahashi; Masanori Yamaguchi; Atushi Kon; Keiichi Takagaki (171-177).
Five isomers with different electric charge were fractionated from human urinary trypsin inhibitor (UTI) by anion exchange HPLC. Intact low-sulfated chondroitin 4-sulfate chains from the isomers were analyzed by HPLC and mass spectrometry. Unsaturated disaccharide composition analysis of the chondroitin sulfate chain revealed that the five isomers differ in the numbers of 4-sulfated disaccharide units. Intriguingly, we detected the presence of multiple novel isomers with different numbers of non-sulfated disaccharide units even in the same charge isomer fraction. Our results demonstrate that UTI can vary in terms of both the degree of sulfation and the length of the low-sulfated chondroitin 4-sulfate chain.
Keywords: Urinary trypsin inhibitor; Glycosaminoglycan; Chondroitin sulfate;

A monoclonal antibody inhibits gelatinase B/MMP-9 by selective binding to part of the catalytic domain and not to the fibronectin or zinc binding domains by Erik Martens; An Leyssen; Ilse Van Aelst; Pierre Fiten; Helene Piccard; Jialiang Hu; Francis J. Descamps; Philippe E. Van den Steen; Paul Proost; Jo Van Damme; Grazia Maria Liuzzi; Paolo Riccio; Eugenia Polverini; Ghislain Opdenakker (178-186).
Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a multidomain enzyme functioning in acute and chronic inflammatory and neoplastic diseases. It belongs to a family of more than 20 related zinc proteinases. Therefore, the discovery and the definition of the action mechanism of selective MMP inhibitors form the basis for future therapeutics. The monoclonal antibody REGA-3G12 is a most selective inhibitor of human gelatinase B. REGA-3G12 was found to recognize the aminoterminal part and not the carboxyterminal O-glycosylated and hemopexin protein domains. A variant of gelatinase B, lacking the two carboxyterminal domains, was expressed in insect cells and fragmented with purified proteinases. The fragments were probed by one- and two-dimensional Western blot and immunoprecipitation experiments with REGA-3G12 to map the interactions between the antibody and the enzyme. The interaction unit was identified by Edman degradation analysis as the glycosylated segment from Trp116 to Lys214 of gelatinase B. The sequence of this segment was analysed by hydrophobicity/hydrophilicity, accessibility and flexibility profiling. Four hydrophilic peptides were chemically synthesized and used in binding and competition assays. The peptide Gly171–Leu187 in molar excess inhibited partially the binding of MMP-9 to REGA-3G12 and thus refines the structure of the conformational binding site. These results define part of the catalytic domain of gelatinase B/MMP-9, and not the zinc-binding or fibronectin domains, as target for the development of selective inhibitors.
Keywords: Gelatinase B; Matrix metalloproteinase-9; Domain mapping; Monoclonal antibody; Hydrophilicity;

Two phospholipase A2 (PLA2) enzymes (NK-PLA2-A and NK-PLA2-B) were purified from the venom of the monocled cobra Naja kaouthia. The molecular weights of NK-PLA2-A and NK-PLA2-B, as estimated by mass spectrometry, were 13,619 and 13,303 Da respectively. Both phospholipases were highly thermostable, had maximum catalytic activity at basic pH, and showed preferential hydrolysis of phosphatidylcholine. Intravenous injection of either PLA2 up to a dose of 10 mg/kg body weight was non-toxic to mice and did not show neurotoxic symptoms. The N. kaouthia PLA2s displayed anticoagulant and cytotoxic activity, but poor hemolytic activity. Both the PLA2s were more toxic to Sf9 and Tn cells compared to VERO cells. NK-PLA2 exhibited selective lysis of wild-type baculovirus-infected Sf9 cells compared to normal cells. Amino acid modification studies and heating experiments suggest that separate sites in the NK-PLA2 molecules are responsible for their catalytic, anticoagulant and cytotoxic activities.
Keywords: Biomembrane; Cytotoxicity; Hemolysis; Membrane phospholipid; Membrane domain; Naja kaouthia phospholipase A2; Sf9 cell; Tn cell; VERO cell;

Mechanistic insights into linear polyethylenimine-mediated gene transfer by Miriam Breunig; Uta Lungwitz; Renate Liebl; Juergen Klar; Birgit Obermayer; Torsten Blunk; Achim Goepferich (196-205).
We recently debuted a variety of linear polyethylenimines (LPEIs) with low molecular weight as carriers for gene delivery. The highest transfection efficiency (∼ 44%) was obtained with LPEI 6.6 kDa, while the cytotoxicity remained low (∼ 90% of CHO-K1 cells survived the transfection procedure). Here, we investigated various steps during the transfection process using LPEI 8.1, 5.0 and 1.8 kDa, in order to gain a more complete insight into LPEI-mediated gene transfer and to explore conceptual aspects for further optimization. The cellular uptake characterized by flow cytometry was similar for LPEI 8.1 and 5.0 kDa, while it was significantly lower for LPEI 1.8 kDa. The transfection efficacy in contrast was at NP 24 20.07% for LPEI 8.1 kDa and 39.71% for LPEI 5.0 kDa. This suggests that the endocytosis seems not to be a decisive parameter that determines the efficacy of a polymer in the transfection process. Real-time PCR investigations revealed that LPEI 1.8 kDa likewise or even better protected plasmid from degradation compared to LPEI 5.0 or 8.1 kDa. Furthermore, we found that 1/6 to 1/3 intact plasmid DNA reached the intracellular compartments after complexation with LPEI 1.8 kDa. Therefore, the amount of plasmid DNA available in the cytoplasm seems not to be a limiting factor in the transfection process. That LPEI 8.1-polyplexes built at NP 12 in glucose and transfected in serum-free culture conditions were superior to those built in sodium chloride or transfected in serum-containing conditions points at the structure as a decisive parameter deserving more attention in future studies.
Keywords: Transfection efficiency; Cytotoxicity; Linear polyethylenimine; Flow cytometry; Confocal laser scanning micrsoscopy; Real-time PCR;

Antigen-mediated growth control of hybridoma cells via a human artificial chromosome by Masahiro Kawahara; Toshiaki Inoue; Xianying Ren; Takahiro Sogo; Hidetoshi Yamada; Motonobu Katoh; Hiroshi Ueda; Mitsuo Oshimura; Teruyuki Nagamune (206-212).
Human artificial chromosome (HAC) vectors possess several characteristics sufficient for the requirements of gene therapy vectors, including stable episomal maintenance and mediation of long-term transgene expression. In this study, we adopted an antigen-mediated genetically modified cell amplification (AMEGA) system employing an antibody/cytokine receptor chimera that triggers a growth signal in response to a cognate non-toxic antigen, and applied it to growth control of HAC-transferred cells by adding an antigen that differed from cytokines that may manifest pleiotropic effects. We previously constructed a novel HAC vector, 21ΔqHAC, derived from human chromosome 21, housed in CHO cells. Here, we constructed an HAC vector harboring an ScFv-gp130 chimera responsive to fluorescein-conjugated BSA (BSA-FL) as well as a model transgene, enhanced green fluorescent protein (EGFP), in CHO cells. The modified HAC was transferred into interleukin (IL)-6-dependent hybridoma 7TD1 cells by microcell-mediated chromosome transfer, and the cells were subsequently found to show BSA-FL-dependent cell growth and sustained expression of EGFP in the absence of IL-6. The AMEGA system in combination with HAC technology will be useful for increasing the efficacy of gene therapy by conferring a growth advantage on the genetically modified cells.
Keywords: Antibody; Receptor; Human artificial chromosome; Growth control; Mammalian cell;

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases by F.A.V. Castro; R.S. Herdeiro; A.D. Panek; E.C.A. Eleutherio; M.D. Pereira (213-220).
Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione–GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.
Keywords: Glutathione transferase; Menadione; Oxidative stress; Saccharomyces cerevisiae;

19F NMR studies of solvent exposure and peptide binding to an SH3 domain by Ferenc Evanics; Julianne L. Kitevski; Irina Bezsonova; Julie Forman-Kay; R. Scott Prosser (221-230).
19F NMR was used to study topological features of the SH3 domain of Fyn tyrosine kinase for both the free protein and a complex formed with a binding peptide. Metafluorinated tyrosine was biosynthetically incorporated into each of 5 residues of the G48M mutant of the SH3 domain (i.e. residues 8, 10, 49 and 54 in addition to a single residue in the linker region to the C-terminal polyhistidine tag). Distinct 19F NMR resonances were observed and subsequently assigned after separately introducing single phenylalanine mutations. 19F NMR chemical shifts were dependent on protein concentration above 0.6 mM, suggestive of dimerization via the binding site in the vicinity of the tyrosine side chains. 19F NMR spectra of Fyn SH3 were also obtained as a function of concentration of a small peptide (2-hydroxynicotinic-NH)–Arg–Ala–Leu–Pro–Pro–Leu–Pro-diaminopropionic acid –NH2, known to interact with the canonical polyproline II (PPII) helix binding site of the SH3 domain. Based on the 19F chemical shifts of Tyr8, Tyr49, and Tyr54, as a function of peptide concentration, an equilibrium dissociation constant of 18 ± 4 μM was obtained. Analysis of the line widths suggested an average exchange rate, k ex, associated with the peptide–protein two-site exchange, of 5200 ± 600 s− 1 at a peptide concentration where 96% of the FynSH3 protein was assumed to be bound. The extent of solvent exposure of the fluorine labels was studied by a combination of solvent isotope shifts and paramagnetic effects from dissolved oxygen. Tyr54, Tyr49, Tyr10, and Tyr8, in addition to the Tyr on the C-terminal tag, appear to be fully exposed to the solvent at the metafluoro position in the absence of binding peptide. Tyr54 and, to some extent, Tyr10 become protected from the solvent in the peptide bound state, consistent with known structural data on SH3–domain peptide complexes. These results show the potential utility of 19F-metafluorotyrosine to probe protein–protein interactions in conjunction with paramagnetic contrast agents.
Keywords: Fluorine NMR; Fyn SH3; Solvent exposure; Oxygen; Paramagnetic effect;

Allosteric inhibition of rat neuronal nitric-oxide synthase caused by interference with the binding of calmodulin to the enzyme by Koji Ohashi; Takeshi Yamazaki; Shigeyuki Kitamura; Shigeru Ohta; Shunsuke Izumi; Shiro Kominami (231-240).
A sigmoid-type dependence on the inhibitor concentration was observed in the cytochrome c reductase activity for peptide inhibitors (mastoparan and melittin), calmodulin antagonists (W-7 and tamoxifen) and monobutyltin in a reconstituted system comprised of recombinant rat neuronal nitric-oxide synthase (nNOS) and calmodulin (CaM). The increase in the concentration of CaM in the system induced a decrease in the inhibitory effect, indicating that the inhibitors might interfere with the interaction between nNOS and CaM. The changes in the fluorescence spectra of dansylated CaM caused by the addition of mastoparan, melittin and monobutyltin indicated complex formation between CaM and those compounds, which led to the decrease in the effective concentration of CaM available to nNOS. The sigmoid-type inhibition of mastoparan and melittin fit the theoretical equations quite well, assuming that two CaM molecules bind cooperatively to one nNOS homodimer. Monobutyltin, tamoxifen and W-7 were found to inhibit nNOS activity by binding to the CaM binding site of the nNOS homodimer, in addition to the binding of the inhibitors to calmodulin. These compounds inhibited the l-citrulline formation of nNOS from l-arginine, and the inhibitory effects were abrogated by raising the concentration of calmodulin. It became clear that the binding of calmodulin to nNOS can be interfered with in two ways: (1) via a decrease in the effective concentration of calmodulin caused by complex formation between the inhibitor and calmodulin, and (2) via the inhibition of the binding of calmodulin to nNOS caused by the occupation of the binding site by the inhibitor.
Keywords: nNOS; Calmodulin; Allosteric inhibition; Homodimer; Melittin; Mastoparan; Calmodulin antagonist;

Analysis of plasma proteins that bind to glycosaminoglycans by Akio Saito; Hiroshi Munakata (241-246).
Glycosaminoglycan-binding proteins, with specific emphasis on dermatan sulfate, have been investigated in human plasma by affinity chromatography, mass spectrometry and Western blotting. Diluted plasma was applied to affinity columns and bound protein was eluted with 500 mM NaCl. Dermatan sulfate and heparan sulfate bound 7% of the total protein. Heparin bound 22% of the total protein, but chondroitin sulfate A bound only 0.23%. Mass spectrometric analysis identified 20 proteins as dermatan-sulfate-binding proteins, most of which were confirmed by Western blotting. Some of these binding proteins, such as fibrinogen, fibronectin, apolipoprotein B, LMW kininogen, inter-α-trypsin inhibitor, and factor H, were degraded to various extents during the chromatography step, but this degradation could be prevented by the inclusion of a serine protease inhibitor. The protein fraction binding to the dermatan sulfate column showed amidase activity, whereas that binding to the heparan sulfate and heparin columns showed 1/2 and 1/20, respectively, of the activity of the dermatan sulfate binding fraction. Dermatan sulfate was similar to heparan sulfate with respect to its capacity to bind plasma proteins and its activation of protease, but differed from chondroitin sulfate and heparin in these properties.
Keywords: Glycosaminoglycan; Dermatan sulfate; Affinity column; Mass spectrometry;

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase by Subhankar Dolai; Rajesh K. Yadav; Alok K. Datta; Subrata Adak (247-256).
We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H2O2 with concurrent evolution of O2. This pseudocatalatic degradation of H2O2 to O2 is solely dependent on ascorbate and is blocked by a spin trap, α-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H2O with subsequent regeneration of ascorbate by the reduction of MDA with H2O2 evolving O2 through the intermediate formation of O2 . Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN. Further kinetic studies indicate that SCN itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H2O2 to O2. Binding studies by optical difference spectroscopy indicate that SCN binds LmAPX (Kd = 100 ± 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.
Keywords: Leishmania; Ascorbate; Peroxidase; Thiocyanate; Pseudocatalase activity;

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus by Nalini S. Mehta; Stéphane L. Benoit; Jagannatha Mysore; Robert J. Maier (257-265).
Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O2 and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O2 exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.
Keywords: Liver disease; Oxidative stress; Catalase; Compensatory mutation; Coagulative necrosis; Chromosomal complementation; KatA gene; Lipid peroxide; Iron sequestering; Hepatitis; Lipid peroxidation;

In vivo effects of chronic contamination with depleted uranium on vitamin D3 metabolism in rat by E. Tissandié; Y. Guéguen; J.M.A. Lobaccaro; L. Grandcolas; P. Voisin; J. Aigueperse; P. Gourmelon; M. Souidi (266-272).
The extensive use of depleted uranium (DU) in today's society results in the increase of the number of human population exposed to this radionuclide. The aim of this work was to investigate in vivo the effects of a chronic exposure to DU on vitamin D3 metabolism, a hormone essential in mineral and bone homeostasis. The experiments were carried out in rats after a chronic contamination for 9 months by DU through drinking water at 40 mg/L (1 mg/rat/day). This dose corresponds to the double of highest concentration found naturally in Finland. In DU-exposed rats, the active vitamin D (1,25(OH)2D3) plasma level was significantly decreased. In kidney, a decreased gene expression was observed for cyp24a1, as well as for vdr and rxrα, the principal regulators of CYP24A1. Similarly, mRNA levels of vitamin D target genes ecac1, cabp-d28k and ncx-1, involved in renal calcium transport were decreased in kidney. In the brain lower levels of messengers were observed for cyp27a1 as well as for lxrβ, involved in its regulation. In conclusion, this study showed for the first time that DU affects both the vitamin D active form (1,25(OH)2D3) level and the vitamin D receptor expression, and consequently could modulate the expression of cyp24a1 and vitamin D target genes involved in calcium homeostasis.
Keywords: Depleted uranium; Cytochrome P450; Vitamin D3; Vitamin D receptor; Chronic contamination;

TRB3 interacts with CtIP and is overexpressed in certain cancers by Jianmin Xu; Shun Lv; Yan Qin; Fang Shu; Yanjuan Xu; Jian Chen; Bing-e Xu; Xiaoqing Sun; Jun Wu (273-278).
TRB3, a human homolog of Drosophila Tribbles, has been recently shown as a critical negative regulator of Akt and S6 kinase activation in a number of cellular processes. Here we found that TRB3 interacted with an important cell cycle regulator CtIP (CtBP-interacting protein) and the interaction involved the C-terminus of both proteins. Interestingly, TRB3 and CtIP co-localized to the nucleus in HeLa cells and exhibited a unique dot-like pattern. Finally, we demonstrated that TRB3 was overexpressed in multiple tumor tissues. Since CtIP plays important roles in cell cycle checkpoint control and it has been implicated in tumorigenesis, our data suggest that TRB3 may be involved in these biological processes through interacting with CtIP.
Keywords: TRB3; CtIP; Tumorigenesis; Co-localization;

In Ramos cells, a human Burkitt's lymphoma cell line, stimulation of the B cell antigen receptor with anti-IgM antibody (Ab) induces apoptosis as indicated by a decrease in cell viability and an increase in DNA fragmentation and cell surface exposure of phosphatidylserine. Furthermore, these changes are suppressed by incubating the cells in α1-acid glycoprotein (AGP)-coated tissue culture plates. Here, we found that, during Anti-IgM Ab-induced apoptosis in Ramos cells, caspase-3 is activated downstream of caspase-8 and the mitochondrial pathway is activated, as indicated by a loss of mitochondrial membrane potential, an increase in the release of cytochrome c to the cytoplasm, and enhanced Bax expression. Anti-IgM Ab-induced apoptosis of neuraminidase-treated Ramos cells was suppressed by incubating the cells on plates coated with AGP, which contains a high concentration of α2,6-linked sialic acid. The incubation on plates coated with AGP also suppressed anti-IgM Ab-stimulated caspase-3 activity and increased the level of X-linked inhibitor of apoptosis protein (XIAP), but it did not affect caspase-8 activity, the mitochondrial membrane potential, cytochrome c release, or Bax expression. The results indicate that the interaction of Ramos cells with immobilized α2,6-linked sialic acid enhances XIAP expression, directly or indirectly suppressing caspase-3 activity and inhibiting anti-IgM Ab-induced apoptosis.
Keywords: α2,6-linked sialic acid; Anti-IgM antibody; Apoptosis; Caspase; XIAP; CD22;

The functional molecular mass of the Pasteurella hyaluronan synthase is a monomer by Philip E. Pummill; Tasha A. Kane; Ellis S. Kempner; Paul L. DeAngelis (286-290).
Hyaluronan (HA), a linear polysaccharide composed of β1,3-GlcNAc-β1,4-GlcUA repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. All known HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The bacterial hyaluronan synthase, PmHAS from Gram-negative Pasteurella multocida, is a 972-residue membrane-associated protein. Previously, the Gram-positive Streptococcus pyogenes enzyme, SpHAS (419 residues), and the vertebrate enzyme, XlHAS1 (588 residues), were found to function as monomers of protein, but the PmHAS is not similar at the protein sequence level and has quite different enzymological properties. We have utilized radiation inactivation to measure the target size of recombinant full-length and truncated PmHAS. The target size of HAS activity was confirmed using internal enzyme standards of known molecular weight. We found that the Pasteurella HA synthase protein functions catalytically as a monomer. Functional truncated soluble PmHAS also behaves as a polypeptide monomer as assessed by gel filtration chromatography and light scattering.
Keywords: Glycosaminoglycan; Glycosyltransferase; Hyaluronan; Hyaluronan synthase; Pasteurella multocida; Radiation inactivation;

GABA-synthesizing enzyme, GAD67, from dermal fibroblasts: Evidence for a new skin function by Kenichi Ito; Kiyotaka Tanaka; Yukinobu Nishibe; Junichi Hasegawa; Hiroshi Ueno (291-296).
Glutamate decarboxylase (GAD) catalyzes the synthesis of γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, from glutamate. An expression of GAD protein has been reported for brain and pancreas, but not for skin. In this study, we present evidence that GAD67 mRNA and protein are expressed in mouse skin and in human dermal fibroblasts. The expression of GAD67 gene is weaker in aged mouse than the young one. To further explore the function of GAD in skin, we have examined a potential role(s) of GABA in human dermal fibroblasts. We have observed that GABA stimulates the synthesis of hyaluronic acid (HA) and enhances the survival rate of the dermal fibroblasts when fibroblasts are exposed to H2O2 an oxidative stress agent. Also observed were lowering the levels of HA and collagen in the embryonic skin from GAD67 deficient mouse as compared to those from the wild-type (WT) mouse. In this study, we have presented the evidences that GAD67 is localized in the dermis and is potentially involved in variety of skin activities.
Keywords: GAD67; GABA; Skin; Dermal fibroblast;

Engineering the sialic acid in organs of mice using N-propanoylmannosamine by Daniel Gagiannis; Reinhart Gossrau; Werner Reutter; Martin Zimmermann-Kordmann; Rüdiger Horstkorte (297-306).
Sialic acids play an important role during development, regeneration and pathogenesis. The precursor of most physiological sialic acids, such as N-acetylneuraminic acid is N-acetyl-d-mannosamine. Application of the novel N-propanoylmannosamine leads to the incorporation of the new sialic acid N-propanoylneuraminic acid into cell surface glycoconjugates. Here we analyzed the modified sialylation of several organs with N-propanoylneuraminic acid in mice. By using peracetylated N-propanoylmannosamine, we were able to replace in vivo between 1% (brain) and 68% (heart) of physiological sialic acids by N-propanoylneuraminic acid. The possibility to modify cell surfaces with engineered sialic acids in vivo offers the opportunity to target therapeutic agents to sites of high sialic acid concentration in a variety of tumors. Furthermore, we demonstrated that application of N-propanoylmannosamine leads to a decrease in the polysialylation of the neural cell adhesion molecule in vivo, which is a marker of poor prognosis for some tumors with high metastatic potential.
Keywords: Sialic acid; Polysialic acid; Neural cell adhesion molecule; Biochemical engineering;

IpaD is localized at the tip of the Shigella flexneri type III secretion apparatus by Musa Sani; Anne Botteaux; Claude Parsot; Philippe Sansonetti; Egbert J. Boekema; Abdelmounaaïm Allaoui (307-311).
Type III secretion (T3S) systems are used by numerous Gram-negative pathogenic bacteria to inject virulence proteins into animal and plant host cells. The core of the T3S apparatus, known as the needle complex, is composed of a basal body transversing both bacterial membranes and a needle protruding above the bacterial surface. In Shigella flexneri, IpaD is required to inhibit the activity of the T3S apparatus prior to contact of bacteria with host and has been proposed to assist translocation of bacterial proteins into host cells. We investigated the localization of IpaD by electron microscopy analysis of cross-linked bacteria and mildly purified needle complexes. This analysis revealed the presence of a distinct density at the needle tip. A combination of single particle analysis, immuno-labeling and biochemical analysis, demonstrated that IpaD forms part of the structure at the needle tip. Anti-IpaD antibodies were shown to block entry of bacteria into epithelial cells.
Keywords: IpaD; Needle complex; Type III secretion system; Shigella flexneri; Electron microscopy;