BBA - General Subjects (v.1760, #9)
Editorial Board (ii).
Short time exposure to hypoxia promotes H9c2 cell growth by Rie Takahashi; Akiyuki Kawawa; Shunichiro Kubota (1293-1297).
The effects of short time (15 min) exposure to hypoxia on rat cardiomyocytes (H9c2) were examined. Exposure to hypoxia inhibited cell death via activation of MEK/extracellular signal-regulated kinase (ERK). Further, exposure to hypoxia promoted cell growth by down-regulation of p27 and phosphorylation of cyclin-dependent kinase 2 (CDK2) and retinoblastoma protein (Rb).
Keywords: Cardiomyocytes; Cell growth; hypoxia; MEK/ERK; Rb;
Protective role of S-adenosyl-l-methionine against hydrochloric acid stress in Saccharomyces cerevisiae by Dipankar Malakar; Anindya Dey; Anil K Ghosh (1298-1303).
S-adenosyl-l-methionine (AdoMet, 1 mM) protects the stationary phase cells of Saccharomyces cerevisiae against the killing effect of acid (10 mM HCl, pH ∼ 2). Both the acid and the acid plus AdoMet treatment for 2 h increased the plasma membrane H+-ATPase activity; thereafter it decreased to the basal level. AdoMet partially recovered the intracellular pH (pHin) that dropped in presence of acid. AdoMet treatment facilitated acid induced phospholipid biosynthesis as well as membrane proliferation, which was reflected in the cellular lipid composition.
Keywords: Saccharomyces cerevisiae; S-Adenosyl-l-methionine; pHin; H+-ATPase; Phosphatidylcholine; Phosphatidylethanolamine;
Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography by Victor Martin Bolanos-Garcia; Owen Richard Davies (1304-1313).
Immobilised metal affinity chromatography (IMAC) is the most widely used technique for single-step purification of recombinant proteins. However, despite its use in the purification of heterologue proteins in the eubacteria Escherichia coli for decades, the presence of native E. coli proteins that exhibit a high affinity for divalent cations such as nickel, cobalt or copper has remained problematic. This is of particular relevance when recombinant molecules are not expressed at high levels or when their overexpression induces that of native bacterial proteins due to pleiotropism and/or in response to stress conditions. Identification of such contaminating proteins is clearly relevant to those involved in the purification of histidine-tagged proteins either at small/medium scale or in high-throughput processes. The work presented here reviews the native proteins from E. coli most commonly co-purified by IMAC, including Fur, Crp, ArgE, SlyD, GlmS, GlgA, ODO1, ODO2, YadF and YfbG. The binding of these proteins to metal-chelating resins can mostly be explained by their native metal-binding functions or their possession of surface clusters of histidine residues. However, some proteins fall outside these categories, implying that a further class of interactions may account for their ability to co-purify with histidine-tagged proteins. We propose a classification of these E. coli native proteins based on their physicochemical, structural and functional properties.
Keywords: Affinity chromatography; E. coli contaminant protein; Metal-binding classification; Histidine-tagged protein; Protein purification;
The anhydrofructose pathway and its possible role in stress response and signaling by Shukun Yu; Roland Fiskesund (1314-1322).
Anhydrofructose (AF) pathway describes the catabolism of α-1,4-glucans of glycogen, starch and maltosaccharides to various metabolites via the central intermediate AF. The reaction sequence of the pathway consists of more than 10 enzymatic steps. This pathway occurs in certain bacteria, fungi, algae and mammals. In this communication, the AF pathway and its regulatory mechanisms in these organisms are presented and the metabolites of this pathway as antioxidants and antimicrobials in biotic and abiotic stress responses and in carbon starvation signaling are discussed.
Keywords: 1,5-Anhydrofructose; Anhydrofructose pathway; Antimicrobial; Antioxidant, Carbon starvation; Glycogen catabolism; Stress response;
Antimicrobial peptides from chilli pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells by Mariângela S.S. Diz; André O. Carvalho; Rosana Rodrigues; Ana Gisele C. Neves-Ferreira; Maura Da Cunha; Elias Walter Alves; Anna L. Okorokova-Façanha; Marco Antônio Oliveira; Jonas Perales; Olga L.T. Machado; Valdirene M. Gomes (1323-1332).
During the last few years, a growing number of cysteine-rich antimicrobial peptides has been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides play an important role in the protection of plants against microbial infection. In this work, proteins from chilli pepper (Capsicum annuum L.) seeds were extracted in phosphate buffer, pH 5.4 and peptides purification were performed by employing ion-exchange chromatographies on DEAE, CM-Sepharose, Sephacryl S-100 and reverse phase in HPLC. Three peptide enriched fractions, namely F1, F2 and F3, were obtained after the CM-Sepharose chromatography. The F1 fraction, mainly composed of three peptides ranging from 6 to 10 kDa, was submitted to N-terminal amino acid sequencing. The closer to 10 kDa peptide showed high sequence homology to lipid transfer proteins (LTPs) previously isolated from others seeds. F1 fraction exhibited strong fungicidal activity against Candida albicans, Saccharomyces cerevisiae and Schizosaccharomyces pombe and also promoted several morphological changes to C. albicans, including the formation of pseudohyphae, as revealed by scanning electron micrography. F1 fraction also reduced the glucose stimulated acidification of the medium mediated by H+-ATPase of S. cerevisiae cells in a dose-dependent manner and caused the permeabilization of yeast plasma membrane to the dye SYTOX Green, as verified by confocal laser microscopy.
Keywords: Lipid transfer protein; Membrane permeabilization; Capsicum annuum; Plant antimicrobial peptide; Saccharomyces cerevisiae; Candida albicans;
Effects of a natural extract from Mangifera indica L, and its active compound, mangiferin, on energy state and lipid peroxidation of red blood cells by Janet Rodríguez; Donato Di Pierro; Magda Gioia; Susanna Monaco; René Delgado; Massimiliano Coletta; Stefano Marini (1333-1342).
Following oxidative stress, modifications of several biologically important macromolecules have been demonstrated. In this study we investigated the effect of a natural extract from Mangifera indica L (Vimang), its main ingredient mangiferin and epigallocatechin gallate (EGCG) on energy metabolism, energy state and malondialdehyde (MDA) production in a red blood cell system. Analysis of MDA, high energy phosphates and ascorbate was carried out by high performance liquid chromatography (HPLC). Under the experimental conditions, concentrations of MDA and ATP catabolites were affected in a dose-dependent way by H2O2. Incubation with Vimang (0.1, 1, 10, 50 and 100 μg/mL), mangiferin (1, 10, 100 μg/mL) and EGCG (0.01, 0.1, 1, 10 μM) significantly enhances erythrocyte resistance to H2O2-induced reactive oxygen species production. In particular, we demonstrate the protective activity of these compounds on ATP, GTP and total nucleotides (NT) depletion after H2O2-induced damage and a reduction of NAD and ADP, which both increase because of the energy consumption following H2O2 addition. Energy charge potential, decreased in H2O2-treated erythrocytes, was also restored in a dose-dependent way by these substances. Their protective effects might be related to the strong free radical scavenging ability described for polyphenols.
Keywords: Antioxidant; EGCG; Mangifera indica L; Mangiferin; Erythrocyte;
Bioelectrocatalytic properties of lignin peroxidase from Phanerochaete chrysosporium in reactions with phenols, catechols and lignin-model compounds by Elena E. Ferapontova; John Castillo; Lo Gorton (1343-1354).
Bioelectrocatalytic reduction of H2O2 catalysed by lignin peroxidase from Phanerochaete chrysosporium (LiP) was studied with LiP-modified graphite electrodes to elucidate the ability of LiP to electro-enzymatically oxidise phenols, catechols, as well as veratryl alcohol (VA) and some other high-redox-potential lignin model compounds (LMC). Flow-through amperometric experiments performed at +0.1 V vs. Ag|AgCl demonstrated that LiP displayed significant bioelectrocatalytic activity for the reduction of H2O2 both directly (i.e., in direct electron transfer (ET) reaction between LiP and the electrode) and using most of studied compounds acting as redox mediators in the LiP bioelectrocatalytic cycle, with a pH optimum of 3.0. The bioelectrocatalytic reduction of H2O2 mediated by VA and effects of VA on the efficiency of bioelectrocatalytic oxidation of other co-substrates acting as mediators were investigated. The bioelectrocatalytic oxidation of phenol- and catechol derivatives and 2,2′-azino-bis(3-ethyl-benzothiazoline-6-sulphonate) by LiP was independent of the presence of VA, whereas the efficiency of the LiP bioelectrocatalysis with the majority of other LMC acting as mediators increased upon addition of VA. Special cases were phenol and 4-methoxymandelic acid (4-MMA). Both phenol and 4-MMA suppressed the bioelectrocatalytic activity of LiP below the direct ET level, which was, however, restored and increased in the presence of VA mediating the ET between LiP and these two compounds. The obtained results suggest different mechanisms for the bioelectrocatalysis of LiP depending on the chemical nature of the mediators and are of a special interest both for fundamental science and for application of LiP in biotechnological processes as solid-phase bio(electro)catalyst for decomposition/detection of recalcitrant aromatic compounds.
Keywords: Lignin peroxidase; Bioelectrocatalysis; Lignin model compound; Veratryl alcohol; 4-Methoxymandelic acid; Phenol; Direct and mediated electron transfer;
Binding of sialyl Lewis X antigen to lectin-like receptors on NK cells induces cytotoxicity and tyrosine phosphorylation of a 17-kDa protein by Koji Higai; Akihiro Ichikawa; Kojiro Matsumoto (1355-1363).
Natural killer (NK) cells mediate cytotoxicity through cell-surface receptors including lectin-like receptors. We have investigated whether sialyl Lewis X (sLeX) antigen, Neu5Acα2,3Galβ1,4(Fucα1,3) GlcNAc-R, can bind to the lectin-like receptors on human NK-derived KHYG cells, using transferrin secreted by human hepatoma-derived HepG2 cells (Hep-TF), whose N-glycans are rich in α1,3-fucosylated bi-, tri-, and tetra-antennary type complexes, and commercially available human transferrin (Nor-TF), which is comprised of bi-antennary N-glycans without α1,3-fucosylation. Results: High sLeX-expressing erythroleukemia-derived K562 cells isolated from fucosyltransferase-3-transfected cells were 2.5-fold more susceptible than wild-type K562 cells to KHYG cells. Fluorescein isothiocyanate (FITC)-labeled Hep-TF bound 1.8-fold more strongly to KHYG cells than did FITC-labeled Nor-TF; the binding was suppressed by treatment with anti-NKG2D, anti-NKG2C, anti-CD94 and anti-CD161 antibodies. FITC-labeled Hep-TF bound more strongly to human monocyte-derived U937 cells transfected with NKG2D and CD94 than to wild-type U937 cells. Moreover, tyrosine phosphorylation of a 17-kDa protein in the KHYG cells was enhanced by incubation on a Hep-TF coated plate and treatment with an anti-NKG2D antibody, but not by a Nor-TF coated plate and an anti-CD94 antibody. Conclusion: The interaction of sLeX antigen with lectin-like receptors on NK cells induces cytotoxicity that is mediated through a tyrosine-phosphorylated 17-kDa protein.
Keywords: Lectin-like receptor; NK cell; NKG2D; CD94; Sialyl Lewis X;
Regulation of mitochondrial morphology and cell survival by Mitogenin I and mitochondrial single-stranded DNA binding protein by Naokatu Arakaki; Takeshi Nishihama; Akira Kohda; Hiroyuki Owaki; Yoshinori Kuramoto; Reika Abe; Toshiyuki Kita; Midori Suenaga; Toshiki Himeda; Masamichi Kuwajima; Hirofumi Shibata; Tomihiko Higuti (1364-1372).
We found that a mouse homolog of human DNA polymerase delta interacting protein 38, referred to as Mitogenin I in this paper, and mitochondrial single-stranded DNA-binding protein (mtSSB), identified as upregulated genes in the heart of mice with juvenile visceral steatosis, play a role in the regulation of mitochondrial morphology. We demonstrated that overexpression of Mitogenin I or mtSSB increased elongated or fragmented mitochondria in mouse C2C12 myoblast cells, respectively. On the other hand, the silencing of Mitogenin I or mtSSB by RNA interference led to an increase in fragmented or elongated mitochondria in the cells, respectively, suggesting that Mitogenin I and mtSSB are involved in the processes of mitochondrial fusion and fission, respectively. In addition, we showed that the silencing of Mitogenin I resulted in an increase in the number of trypan blue-positive cells and the silencing of mtSSB resulted in an enhancement of the sensitivity of the cells to apoptotic stimulation by etoposide. The present results demonstrated that these proteins play a role in cell survival.
Keywords: JVS; mtSSB; PDIP38; Mitochondria; Fission/fusion; Mitochondrial biogenesis; Apoptosis;
Indirect oxidation of amino acid phenylhydrazides by mushroom tyrosinase by Beata Gąsowska; Bożena Frąckowiak; Hubert Wojtasek (1373-1379).
We have investigated oxidation of amino acid phenylhydrazides by mushroom tyrosinase in the presence of 4-tert-butylcatechol and N-acetyl-l-tyrosine. Spectrophotometric measurements showed gradual disappearance of 4-tert-butyl-o-benzoquinone, generated by oxidation of 4-tert-butylcatechol with sodium periodate, after addition of amino acid phenylhydrazides. However, the presence of the phenylhydrazides did not influence the concentration of 4-tert-butyl-o-benzoquinone formed during enzymatic oxidation. Oxygen consumption measurements demonstrated that in a mixture both compounds were oxidized but the reaction rate was proportional to the concentration of the catechol. In the oxidation of N-acetyl-l-tyrosine addition of phenylhydrazides shortened the lag period, indicating that they acted as reducing agents, converting N-acetyl-l-dopaquinone to N-acetyl-l-dopa. In HPLC analysis of the oxidation 4-tert-butylcatechol and the phenylhydrazide of Boc-tryptophan only the N-protected amino acid and 4-tert-butyl-o-benzoquinone were detected as final products. In the presence of the natural substrates the oxidation of amino acid phenylhydrazides required much smaller amounts of the enzyme and was up to 40 times faster than the reaction carried out without these compounds. These results demonstrate that tyrosinase can oxidize phenylhydrazides indirectly through o-quinones. This reaction explains the inhibitory effect of agaritine, a natural amino acid hydrazide, on melanin formation and the inhibitory effects of other hydrazine derivatives on tyrosinase described in the literature.
Keywords: Tyrosinase; o-quinone; Hydrazine; Hydrazide; Agaritine; Redox exchange;
Perchloric acid-soluble protein is expressed in enterocytes and goblet cells in the intestine and upregulated by dietary lipid by Hiroaki Kanouchi; Mami Miyamoto; Tatsuzo Oka; Mitsuharu Matsumoto; Takeaki Okamoto; Shigenobu Tone; Yohsuke Minatogawa (1380-1385).
We previously identified perchloric acid-soluble protein (PSP) in the rat liver, kidney, brain and lung, and reported that it appeared to be related to repression of cell proliferation. In the present study, we clarified that PSP was expressed in the intestine, and found that the amino acid sequence of the intestinal PSP was consistent with those of other PSPs present in other tissues. An immunohistochemical study revealed that PSP was expressed in enterocytes and goblet cells, but not in other cell types among the lamina propria epithelial cells. A comparison of the expressions of PSP and proliferating cell nuclear antigen demonstrated that the proliferating cells did not express PSP. Intestinal PSP expression was induced by ∼ 3-fold by oral administration of dietary fat. These findings indicate that the proliferation repression activity may be related to renewal of the intestinal epithelium, and that PSP is one of the fatty acid-inducible proteins.
Keywords: Perchloric acid-soluble protein; Rat intestine; Fatty acid-binding protein; Cell proliferation;
Immunocytochemical localization of scorpion digestive lipase by Nacim Zouari; Alain Bernadac; Nabil Miled; Tarak Rebai; Alain De Caro; Souad Rouis; Frederic Carriere; Youssef Gargouri (1386-1392).
The scorpion hepatopancreas consists of digestive diverticula and interstitial tissue. A digestive diverticulum is composed of two differentiated cell types: the secretory zymogene-like cells and the digestive cells which are the most abundant. The scorpion digestive lipase (SDL) has been previously purified from scorpion hepatopancreas, but its cellular localization has not yet been established. Polyclonal antibodies specific to SDL were prepared and used in immunofluorescence and immunogold techniques to determine the cellular location of SDL. Our results clearly established that SDL was detected intracellularly in specific vesicles tentatively named (SDL+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion might occur in specific granules inside the digestive cells, as suggested by previous studies on the scorpion digestive process.
Keywords: Scorpion hepatopancreas; Zymogene-like cell; Digestive cell; Digestive lipase; Immunocytolocalization; Intracellular digestion;
Sialilated β1,6 branched N-oligosaccharides modulate adhesion, chemotaxis and motility of melanoma cells: Effect on invasion and spontaneous metastasis properties by B.V.V.G. Reddy; Rajiv D. Kalraiya (1393-1402).
B16BL6 cells, selected specifically for invasive characteristics from B16F10 mouse melanoma cells, displayed greater ability to metastasize to lungs and produced larger colonies than the parent cells, when injected intravenously. When the two cell lines were compared for surface β1,6-branched N-oligosaccharides by flow cytometry using Leuco–Phyto–Heam–Agglutinin, B16BL6 were found to express significantly higher levels. Inhibition of the oligosaccharide expression, by treatment of the cells with swainsonine or antisense-N-acetyl glucosaminyl-transferase-V, significantly reduced metastasis and invasion (>50%). Further, inhibition of oligosaccharides on the molecules like β1 integrin (one of the major carriers) caused 30–45% reduction in their adherence to extra-cellular-matrix components especially collagen IV and laminin, and chemotaxis towards fibronectin and matrigel. The inhibition also decreased haptotaxis by ∼50% to fibronectin but surprisingly was enhanced towards laminin by ∼75%. The cells on which the expression of these oligosaccharides was inhibited failed to exhibit the characteristic spontaneous metastasis and adhesion properties of B16BL6 cells. In none of the cases, however, the secretion of matrix-metallo-proteases correlated with oligosaccharide expression. Sialylation of surface oligosaccharides was found to be accompanied by even higher motility and adherence to the substrates. These results strongly support an important role of cell surface β1,6-linked N-oligosaccharides, especially the sialylated derivatives, in the processes that influence invasion and metastasis.
Keywords: Adhesion; β1,6branched N-oligosaccharide; Invasion; Motility; Metastasis;
Enhancement of cell attachment and tissue integration by a IKVAV containing multi-domain peptide by X. Lin; K. Takahashi; Y. Liu; P.O. Zamora (1403-1410).
Laminin contains a number of cell binding motifs including IKVAV and some that bind heparin. We developed a multi-domain synthetic peptide, LA2, which combines IKVAV sequences with a heparin-binding domain with the goal of improving cell attachment to otherwise non-adherent substrates. LA2 was used to coat polystyrene, ethyl vinyl acetate (EVA), expanded polytetrafluoroethylene (ePTFE), polycarbonate, titanium and stainless steel. In cell attachment studies, LA2 dramatically increased cell attachment to polystyrene and EVA compared to uncoated counterparts or those coated with SIKVAV. Similar increases were observed on ePTFE and titanium. On polystyrene, LA2 enhanced the attachment of endothelial cells, smooth muscle cells, epithelial cells, myoblasts, and osteoblast progenitor cells. Following adhesion, the cells underwent proliferation to form confluent monolayers with phenotypic morphologies. Using osteoblast progenitor cells (MC3T3 cells) grown on LA2/polystyrene, the cells exhibited an increased production of a differentiation marker, alkaline phosphatase. In vivo, LA2 improved tissue integration into ePTFE when implanted subcutaneously in rats. After 2 weeks, cells had penetrated deep into the LA2 coated ePTFE implant whereas little cell penetration was found in uncoated grafts. The implant sites exhibited little inflammation or other untoward effects. The results indicated that the LA2 peptide improved cell adhesion and tissue integration and might be useful in a number of tissue engineering applications.
Keywords: Synthetic peptide; Laminin; SIKVAV; Cell attachment; ePTFE; Tissue integration;
Sodium orthovanadate induced tyrosine phosphorylation of platelet nitric oxide synthase negatively regulates enzyme activity by Andrew D. Milward; Rocio Riba; Bilal Patel; Nikolaus G. Oberprieler; Khalid M. Naseem (1411-1417).
The post-translational regulation of platelet nitric oxide synthase (NOS) activity is poorly understood. In the present study we examined how tyrosine phosphorylation of NOS, induced by the tyrosine phosphatase inhibitor sodium orthovanadate (VO4), influenced enzyme activity. Platelet NOS was basally tyrosine phosphorylated, but incubation with VO4 (100–1000 μM) led to a concentration-dependent increase in tyrosine phosphorylation of the enzyme with maximal effects observed at 500 μM. Importantly, we observed no change in serine1179 or threonine497 phosphorylation. The increased tyrosine phosphorylation was associated with reduced NOS activity and NO bioavailability, as evidenced by measurement of [3H]-l-citrulline and cGMP respectively. The signalling events underlying the effects of VO4 were studied using specific inhibitors to kinases that are known to influence NOS activity. Preincubation of platelets with the Src kinase inhibitor PP2 (20 μM) blocked VO4-induced tyrosine phosphorylation of NOS and abolished the effects of VO4 on cGMP formation. The PKC inhibitor Ro-31-8220 (10 μM) had no effect on VO4-induced tyrosine phosphorylation, but did have a modest but significant effect on cGMP formation. In contrast, the PI-3-kinase inhibitor wortmannin (100 nM) had no effect on either tyrosine phosphorylation or cGMP formation. Our data indicate that tyrosine phosphorylation may act to repress NOS activity. Furthermore, VO4 induces a Src-dependent, and to a lesser degree PKC-dependent, inhibition of platelet NOS.
Keywords: Platelet; Nitric oxide synthase; cGMP; Tyrosine phosphorylation; Src kinase;
Structure–activity relationships of some hederagenin diglycosides: Haemolysis, cytotoxicity and apoptosis induction by Martin Chwalek; Nathalie Lalun; Hélène Bobichon; Karen Plé; Laurence Voutquenne-Nazabadioko (1418-1427).
Hederagenin saponins are largely represented in nature and possess many biological activities such as haemolytic, antiviral, fungicidal, molluscicidal or cytotoxic, partially due to their interaction with the cell membrane. The lysis of erythrocytes (haemolysis) is a simple test to evaluate this adsorption, and this activity has been linked to the structure of the aglycone and also depends on the sugar moiety of the saponin. To further complete our study of the structure–activity relationships of triterpenoid saponins, α-hederin and related hederagenin diglycosides were synthesized to better understand the influence of the second sugar (α-l-rhamnose, β-d-xylose or β-d-glucose) and the substitution of this sugar on α-l-arabinose (position 2, 3 or 4). Haemolysis and cytotoxic activity on KB cells were tested. These compounds probably interact with membrane cholesterol and produce destabilization of the membrane inducing haemolysis. Cytotoxicity could involve the same mechanism, although some saponins induce an apoptotic process. The nuclear structure of the KB cell was thus investigated by confocal microscopy. The cytotoxic activity of a second group of hederagenin glucoside saponins was also evaluated. Our results showed that cytotoxicity was a result of both the sugar part and the structure of genin (carboxylic acid or methyl ester).
Keywords: α-Hederin; Saponin; Hederagenin; Haemolysis; Cytotoxicity; KB cell;
Drosophila Ecp is a novel ribosome associated protein interacting with dRPL5 by Daoyong Wang; Min Xu; Jiabin Liu; Yongqi Wan; Haiteng Deng; Fei Dou; Wei Xie (1428-1433).
Drosophila Ecp is a phylogenetically conserved protein with homologs in most eukaryotes. Studies on Ecp homologs in rat and human suggest those proteins might be involved in cell cycle control, cell proliferation, and learning and memory. However, the molecular function of Ecp itself remains unclear. We show that both the mRNA and protein of ecp are ubiquitously expressed during the entire fly embryogenesis and life cycle. Results of co-immunoprecipitation show that Ecp forms a stable complex with many ribosomal proteins, including dRPL5. The binding of Ecp to dRPL5 was confirmed by GST pulldown. Furthermore, Ecp was found to cosediment with ribosome subunits in a sucrose gradient. These results indicate that Ecp might be a novel ribosome associated protein interacting with dRPL5.
Keywords: Protein synthesis; Translation initiation; eIFs; Ecp/eIF5C;
Biochemical limitations to high-level expression of humanized monoclonal antibodies in transgenic maize seed endosperm by R. David Law; Douglas A. Russell; Lisa C. Thompson; Sheryl C. Schroeder; Christina M. Middle; Mary T. Tremaine; Thomas P. Jury; Xavier Delannay; Steven C. Slater (1434-1444).
Transgenic plants are potentially valuable systems for the large scale manufacture of therapeutic proteins. To improve this technology, determining the importance of transgene transcript levels on protein accumulation in sink tissues during their development is crucial. In transgenic maize (Zea mays L.) plants expressing humanized monoclonal antibodies (mAbs) in their seed endosperm, steady-state κ light chain (LC) and γ heavy chain (HC) mRNA levels were quantified during development and compared to the levels of fully-assembled mAb protein present at seed maturity. RNA blots and non-reducing SDS-PAGE western immunoblots revealed that steady-state LC and HC mRNA and protein levels were undetectable at 10 days after pollination (DAP) but increased quickly thereafter in three transgenic events expressing different mAb molecules. Similar to γ-zein mRNA, LC and HC messages were highly abundant between 15 and 25 DAP. Quantitative RNA blots and western immunoblots showed that steady-state LC transcript levels during development correlated extremely closely with protein levels in mature seed (r 2 = 0.99). For HC, this correlation was not as strong (r 2 = 0.85). Consistent with this finding, concomitantly increasing the zygosity levels of the LC and HC transgenes enhanced mAb concentration in mature seed, in contrast to increasing the copy number of the transgene insert, which did not correlate with high seed mAb levels. The results indicate that high-level expression of fully-assembled mAb protein in maize endosperm was favored by high LC and HC mRNA levels and was largely limited by HC protein concentration.
Keywords: Expression profiling; Maize endosperm; Maize seed development; Monoclonal antibody; Plant-made pharmaceutical; Transgenic seed;
PIN inhibits nitric oxide and superoxide production from purified neuronal nitric oxide synthase by Yong Xia; Carlos O. Berlowitz; Jay L. Zweier (1445-1449).
A protein inhibitor of neuronal nitric oxide synthase (nNOS) was identified and designated as PIN. PIN was reported to inhibit nNOS activity in cell lysates through disruption of enzyme dimerization. However, there has been lack of direct characterization of the effect of PIN on NO production from purified nNOS. Furthermore, nNOS also generates superoxide (•O2 −) at low levels of l-arginine. It is unknown whether PIN affects •O2 − generation from nNOS. Therefore, we performed direct measurements of the effects of PIN on NO and •O2 − generation from purified nNOS using electron paramagnetic resonance spin trapping techniques. nNOS was isolated by affinity chromatography and a fusion protein CBP-PIN was used to probe the effect of PIN. While the tag CBP did not affect nNOS activity, CBP-PIN caused a dose-dependent inhibition on both NO and l-citrulline production. In the absence of l-arginine, strong •O2 − generation was observed from nNOS, and this was blocked by CBP-PIN in a dose-dependent manner. With low-temperature polyacrylamide gel electrophoresis, neither CBP nor CBP-PIN was found to affect nNOS dimerization. Thus, these results suggested that PIN not only inhibits NO but also •O2 − production from nNOS, and this is through a mechanism other than decomposition of nNOS dimers.
Keywords: PIN; Nitric oxide; Nitric oxide synthase; Superoxide; Dimerization;
Expression of recombinant human antibody fragments capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom by M.B. Tamarozzi; S.G. Soares; S. Marcussi; J.R. Giglio; J.E. Barbosa (1450-1457).
Phospholipases A2 are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. This study used a large nonimmune human scFv library named Griffin.1 (MRC, Cambridge, UK) for selection of recombinant antibodies against antigens present in Bothrops jararacussu venom and identification of specific antibodies able to inhibit phospholipase activity. Four clones were identified as capable of inhibiting this activity in vitro. These clones were able to reduce in vivo the myotoxic activity of BthTX-I and BthTX-II PLA2, but had no effect on the in vitro anticoagulant activity of BthTX-II. This work shows the potential of using recombinant scFv libraries in the search for antibodies that neutralize relevant venom components.
Keywords: Phage Display; scFv; Phospholipase A2; Bothropic venom; Myotoxin;
Mutational analysis of N-glycosylation recognition sites on the biochemical properties of Aspergillus kawachii α-l-arabinofuranosidase 54 by Takuya Koseki; Yozo Miwa; Yuichiro Mese; Akimasa Miyanaga; Shinya Fushinobu; Takayoshi Wakagi; Hirofumi Shoun; Hiroshi Matsuzawa; Katsumi Hashizume (1458-1464).
A role for N-linked oligosaccharides on the biochemical properties of recombinant α-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn83–Thr–Thr and Asn202–Ser–Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn83, Asn202, and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn202 may contribute to thermostability and catalysis.
Keywords: α-l-arabinofuranosidase 54; N-glycosylation; mutational analysis; Aspergillus kawachii;
The vascular endothelial growth factor VEGF165 induces perlecan synthesis via VEGF receptor-2 in cultured human brain microvascular endothelial cells by Toshiyuki Kaji; Chika Yamamoto; Mami Oh-i; Yasuyuki Fujiwara; Yasuo Yamazaki; Takashi Morita; Anna H. Plaas; Thomas N. Wight (1465-1474).
A member of the vascular endothelial growth factor (VEGF) family, VEGF165, regulates vascular endothelial cell functions in autocrine and paracrine fashions in microvessels. Proteoglycans are highly glycosylated poly-anionic macromolecules that influence cellular behaviors such as proliferation and migration by interacting with cytokines/growth factors. In the present study, we investigated the regulation of proteoglycan synthesis by VEGF165 in cultured human brain microvascular endothelial cells. The cells were exposed to recombinant human VEGF165, and the proteoglycans were then characterized using biochemical techniques. VEGF165 treatment increased the accumulation of proteoglycans 1.4- and 1.6-fold in the cell layer and conditioned medium, respectively. This effect resulted from the activation of VEGFR-2, and was mimicked by vammin, a VEGFR-2 ligand from snake venom but not placenta growth factor, which binds specifically to VEGFR-1. VEGF165 stimulated the production and secretion of perlecan, substituted with shorter heparan sulfate side chains, but with unaltered sulfated disaccharide composition. The perlecan secreted by VEGF165-stimulated endothelial cells may be involved in the regulation of cellular behavior during angiogenesis, in diseases of the brain microvessels, and in the maintenance of the endothelial cell monolayer.
Keywords: Vascular endothelial growth factor; Extracellular matrix; Perlecan; Proteoglycan; Heparan sulfate; Endothelial cell;