BBA - General Subjects (v.1722, #1)

Differential influence of bacitracin on plant proteolytic enzyme activities by Marek Radłowski; Sławomir Bartkowiak; Krystyna Winiarczyk; Andrzej Kalinowski (1-5).
The effect of bacitracin on the activity of proteases extracted from pollen and sprouts of various plant species and compared to five commercially available proteases was studied. Bacitracin stimulates some pollen proteolytic enzyme activities, contrary to its inhibitory influence on proteases from the other sources. Proteases from maize pollen, inhibited by pepstatin and phenylmethylsulfonyl fluoride, immediately accelerate their activities after addition of bacitracin to the reaction mixture. The stimulating influence of peptide antibiotic on pollen proteases of some plants is unexpected and molecular mechanism of this phenomenon requires a further elucidation. The augmentation of allergenic response caused by pollen enzymes and drugs containing bacitracin is discussed.
Keywords: Pollen; Protease; Bacitracin; Stimulation of activity;

Rat Sertoli cells express epithelial but also mesenchymal genes after immortalization with SV40 by Lutz Konrad; Marcel Munir Keilani; Andrea Cordes; Elke Völck-Badouin; Leslie Laible; Martin Albrecht; Heiner Renneberg; Gerhard Aumüller (6-14).
A new immortal Sertoli cell line from pubertal rat testis was established and characterized. We have generated the clonal line SCIT-C8 expressing established markers for Sertoli cells (SC) like transferrin, clusterin and steel factor/stem cell factor (SCF). Additionally, the immortalized cells express afadin, a protein which is a member of tight and adherens junctions, therefore the cells may be useful for studies of the blood–testis barrier (BTB) in vitro. In contrast to primary SC, the immortalized cells lost expression of androgen receptor and responsiveness to androgens and follicle-stimulating hormone. Surprisingly, we found mRNA expression and protein secretion of the mesenchymal markers, fibronectin and entactin-1, which we also observed for the immortalized SC lines, ASC-17D and 93RS2. In comparison to primary SC, the immortalized cells demonstrated enhanced adhesion in vitro. This correlated with the expression of entactin-1 because adhesion was strongly reduced by antibody perturbation experiments. Additionally, we found the alternatively spliced and primarily muscle cell-specific long variant of TGF-β2 not only in peritubular cells (PC), but also in the primary and immortalized SC. Furthermore, all immortalized cell lines secreted higher amounts of TGF-β2 than primary SC. In conclusion, the immortalized SC lines from different developmental stages showed a similar pattern of epithelial and mesenchymal markers.
Keywords: Immortalization; ECM protein; Cell adhesion; TGF-β2; Afadin;

Binding of the bioactive component Jatrorrhizine to human serum albumin by Ying Li; Wenying He; Jiaqin Liu; Fenling Sheng; Zhide Hu; Xingguo Chen (15-21).
The interaction between Jatrorrhizine with human serum albumin (HSA) were studied by fluorescence quenching technique, circular dichroism (CD) spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy. Fluorescence data revealed the presence of a single class of binding site on HSA and its binding constants (K) are 7.278×104, 6.526×104, and 5.965×104 L·mol−1 at 296, 303, and 310 K, respectively. The CD spectra and FT-IR spectra have proved that the protein secondary structure changed in the presence of Jatrorrhizine in aqueous solution. The effect of common ions on the binding constants was also investigated. In addition, the thermodynamic functions standard enthalpy (ΔH 0) and standard entropy (ΔS 0) for the reaction were calculated to be −10.891 kJ·mol−1 and 56.267 J·mol−1 K−1, according to the van't Hoff equation. These data indicated that hydrophobic and electrostatic interactions played a major role in the binding of Jatrorrhizine to HSA. Furthermore, the displacement experiments indicated that Jatrorrhizine could bind to the site I of HSA, which was also in agreement with the result of the molecular modeling study.
Keywords: Jatrorrhizine; Human serum albumin; Binding constant; Fluorescence quenching; FT-IR spectroscopy;

Water-soluble low-molecular weight chitosan (LMWC) and chitooligosaccharides (COs) were obtained from chitosan (16% N-acetylation) by depolymerization induced by potassium persulfate under nitrogen atmosphere for 2 h. They were characterized by IR, X-ray, HPLC and 13C-NMR. Splitting of C3/C5 signals in the latter indicated a newer conformation, and also showed prominence of acetyl groups in LMWC, may be due to cleavage between two consecutive deacetylated residues. Molecular weight of LMWC, determined by HPSEC, showed a single peak of ∼37 kDa. HPLC analysis of the solvent-extracted fraction revealed COs enriched with pentamer, hexamer and higher oligomers. The effect of LMWC and COs on the growth of Ehrlich ascites tumor (EAT) cells and tumor-induced neovascularization was studied. COs (50 μg) were more effective compared to LMWC (100 μg) and proved to be potent angioinhibitory and antitumor compounds, as shown by inhibition of angiogenesis and inducing apoptosis as a function of DNA fragmentation.
Keywords: Low molecular weight chitosan; Chitooligosaccharide; Anti-angiogenesis; Apoptosis; DNA;

Formation of oxygen radicals during reduction of H2O2 or diperoxovanadate with vanadyl sulfate or ferrous sulfate was indicated by the 1:2:2:1 electron spin resonance (ESR) signals of the DMPO adduct typical of standard OH radical. Signals derived from diperoxovanadate remained unchanged in the presence of ethanol in contrast to those from H2O2. This gave the clue that they represent a different radical, possibly OV(O2)2+, formed on breaking a peroxo-bridge of diperoxovanadate complex. The above reaction mixtures evolved dioxygen or, when NADH was present, oxidized it rapidly which was accompanied by consumption of dioxygen. Operation of a cycle of peroxovanadates including this new radical is suggested to explain these redox activities both with vanadyl and ferrous sulfates. It can be triggered by ferrous ions released from cellular stores in the presence of catalytic amounts of peroxovanadates.
Keywords: Peroxovanadate cycle; Diperoxovanadate; Vanadyl; OV(O2)2+ radical;

Purification and cDNA cloning of Luxuriosin, a novel antibacterial peptide with Kunitz domain from the Longicorn Beetle, Acalolepta luxuriosa by Kenjiro Ueda; Ayaka Saito; Morikazu Imamura; Nami Miura; Shogo Atsumi; Hiroko Tabunoki; Ayako Watanabe; Madoka Kitami; Ryoichi Sato (36-42).
We have purified a novel antibacterial peptide from the hemolymph of the coleopteran insect Acalolepta luxuriosa, of the family Cerambyocidae, and named it luxuriosin. This peptide showed growth-inhibitory activity against Micrococcus luteus and germination- and/or growth-inhibitory activity against the conidia from rice blast fungus, Magnaporthe grisea. The amino acid sequence determined by cDNA cloning identified luxuriosin as a peptide of 88 amino acids with a theoretical molecular weight of 10,368.34, containing a Kunitz domain.
Keywords: Antimicrobial peptide; Antibacterial peptide; Antifungal peptide; Kunitz; Acalolepta luxuriosa; Insect immunity;

The effect of sub-lethal ALA-PDT on the cytoskeleton and adhesion of cultured human cancer cells by Anatoly Uzdensky; Elona Kolpakova; Asta Juzeniene; Petras Juzenas; Johan Moan (43-50).
5-Aminolevulinic acid (ALA), a precursor of the endogenous photosensitizer protoporphyrin IX, is used in the photodynamic therapy (PDT) of cancer. Sub-lethal ALA-PDT (1-min irradiation with 370–450 nm blue light, 0.6 mW/cm2 after 2-h incubation with 1 mM ALA) has been earlier shown to change cell morphology and to inhibit both trypsin-induced detachment of cultured cancer cells from the plastic substrata and cell attachment to the bottom of the plastic well plates. In the present study, we found that such treatment of human adenocarcinoma WiDr cells grown in dense colonies stimulated the formation of actin cortex between cells in the colonies and increased the number of actin stress fibres in some, but not in all, cells. However, ALA-PDT did not change the microtubular cytoskeleton in these cells. A similar treatment of glioblastoma D54Mg cells, which grow separately and communicate by protrusions, caused loss of fibrillar actin structures in growth cones, retraction of protrusions, and surface blebbing in some cells. The application of the cytoskeleton inhibitors cytochalasin D, colchicine or taxol showed that the inhibition of trypsin-induced detachment of photosensitized WiDr cells was related to ALA-PDT-induced changes in actin and microtubular cytoskeleton. Some signal transduction processes are suggested to be involved in ALA-PDT-induced changes in cytoskeleton, cell shape, and adhesion.
Keywords: 5-Aminolevulinic acid-photodynamic therapy; Cell adhesion; Actin; Tubulin;

The lipophilic dye merocyanine 540 (MC540) localizes primarily in the plasma membrane (PM) of tumor cells, where it can sensitize lethal photoperoxidative damage of potential therapeutic importance. We postulated (i) that chain peroxidation triggered by iron-catalyzed turnover of nascent hydroperoxides (LOOHs) generated by singlet oxygen (1O2) attack on PM lipids contributes significantly to overall cytolethality, and (ii) that nitric oxide (NO), a known scavenger of organic free radicals, would suppress this and, thus, act cytoprotectively. In accordance, irradiation of MC540-sensitized L1210 cells produced 5α-OOH, a definitive 1O2 adduct of PM cholesterol, which decayed during subsequent dark incubation with appearance of other signature peroxides, viz. free-radical-derived 7α/β-OOH. Whereas chemical donor (SPNO or SNAP)-derived NO had little or no effect on post-irradiation 5α-OOH disappearance, it dose-dependently inhibited 7α/β-OOH accumulation, consistent with interception of chain-carrying radicals arising from one-electron reduction of primary LOOHs. Using [14C]cholesterol as an L1210 PM probe, we detected additional after-light products of chain peroxidation, including diols (7α-OH, 7β-OH) and 5,6-epoxides, the yields of which were enhanced by iron supplementation, but strongly suppressed by NO. Correspondingly, photoinitiated cell killing was significantly inhibited by NO introduced either immediately before or after light exposure. These findings indicate that prooxidant LOOH turnover plays an important role in photokilling and that NO, by intercepting propagating radicals, can significantly enhance cellular resistance.
Keywords: Merocyanine 540; Photosensitization; Lipid peroxidation; Nitric oxide; Free radical;

Enzymatic synthesis of GlcNAc-terminated poly-N-acetyllactosamine β-glycosides GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3) n Galβ1,4GlcNAcβ-pNP (n=1–4) was demonstrated using a transglycosylation reaction of Escherichia freundii endo-β-galactosidase. The enzyme catalyzed a transglycosylation reaction on GlcNAcβ1,3Galβ1,4GlcNAcβ-pNP (1), which served both as a donor and an acceptor, and converted 1 into p-nitrophenyl β-glycosides GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)1Galβ1,4GlcNAcβ-pNP (2), GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)2Galβ1,4GlcNAcβ-pNP (3), GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)3Galβ1,4GlcNAcβ-pNP (4) and GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)4Galβ1,4GlcNAcβ-pNP (5). When 2 was used as an initial substrate, it led to the preferential synthesis of nonasaccharide β-glycoside 4 to heptasaccharide β-glycoside 3. This suggests that 4 is directly synthesized by transferring the tetrasaccharide unit GlcNAcβ1,3Galβ1,4GlcNAcβ1,3Gal to nonreducing end GlcNAc residue of 2 itself. The efficiency of production of poly-N-acetyllactosamines by E. freundii endo-β-galactosidase was significantly enhanced by the addition of BSA and by a low-temperature condition. Resulting 2 and 3 were shown to be useful for studying endo-β-galactosidase-catalyzed hydrolytic and transglycosylation reactions.
Keywords: Endo-β-galactosidase; Enzymatic synthesis; Glycosylation; Poly-N-acetyllactosamine; Transglycosylation;

The binding of copper ions to glycine-rich proteins (GRPs) from Cicer arietinum by Masakatsu Kamiya; Yasuhiro Kumaki; Katsutoshi Nitta; Takeshi Matsumoto; Kunio Hikichi; Norio Matsushima (69-76).
Cicer arietinum GRP1 and GRP2 are rich in glycine interposed with histidine and tyrosine. In order to study whether or not these proteins bind Cu2+, circular dichroism (CD) and nuclear magnetic resonance (NMR) were measured for three synthetic peptides corresponding to sections of the protein's sequences including 1, N1Y2G3H4G5G6G7N8Y9G10N11, where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH2 group at the C-terminus. The visible CD spectra for 1 showed a positive peak near 590 nm not at pH 6.0 but pH 7.4 in the presence of copper ions. The Cu2+ binding induced a drastic change in the far-UV CD spectra, showing the occurrence of large conformation changes. In the 2D TOCSY NMR spectra at pH 7.4, the addition of small amounts of CuSO4 caused a significant broadening of proton resonances of not only His4 but also Gly5, Asn8 and Asn11. CD titration experiment suggested that NYGHGGGNYGN including one repeat unit comprises the fundamental Cu2+ binding unit.
Keywords: Glycine-rich protein; Copper binding; Histidine/glycine/tyrosine-rich domain; CD; NMR;

Evaluation of MUC6 mucin tandem repeats by Simon Parry; Mark Sutton-Smith; Paul Heal; Shih-Hsing Leir; Timea Palmai-Pallag; Howard R. Morris; Michael A. Hollingsworth; Anne Dell; Ann Harris (77-83).
The MUC6 mucin was originally isolated from stomach mucus and is one of the major secreted mucins of the digestive tract. A full-length cDNA has not been isolated for this large molecule (greater than 15 kb) and it remains poorly studied. To circumvent the lack of reagents for investigating MUC6, we isolated a cDNA clone from a human fetal pancreatic duct cDNA library that encodes 282 amino acids of the MUC6 tandem repeat. A blast search with the sequence of this cDNA clone showed 90% homology with the original MUC6 (L07517) derived from a human stomach cDNA library and 95% homology both with AK096772, a MUC6-related protein isolated from a human prostate cDNA library and the human genome project clone AC083984. The MUC6 partial cDNA clone isolated from fetal pancreas was inserted into an epitope-tagged MUC1 mucin molecule in place of the native tandem repeat. This chimeric mucin was expressed in human pancreatic (Panc1) and colon (Caco2) carcinoma cell lines and purified for analysis of O-glycosylation by fast atom bombardment mass spectrometry (FAB-MS). The FAB-MS spectra showed O-glycans that had been detected previously on chimeric mucins carrying different tandem repeats, though the spectra for MUC1F/6TR mucins expressed in the Panc1 and Caco2 cells were very different. There was a paucity of O-glycosylation in Panc1 cells in comparison to Caco2 cells where many more structures were evident, and the most abundant glycans in Panc1 cells were sialylated.
Keywords: MUC6 mucin; Pancreas; Panc1 cell; Caco2 cell; Fast atom bombardment mass spectrometry;

The antioxidant effects of natural vitamin B6 compounds on Schizosaccharomyces pombe cells treated with menadione sodium bisulfite (water-soluble menadione and a generator of superoxide, MSB) and the mechanism underlying the function were examined with the yeast cells treated with pyridoxal 5′-phosphate. Vitamin B6 compounds showed no ex vivo reactivity toward MBS at pH 5.5 or 7.0. The yeast cells showed no growth in the medium containing 1.0 mM MSB. The coexistence of 1.0 mM of each vitamin B6 compound supported the growth of the yeast cells. The efficacy order was pyridoxal 5′-phosphate≥pyridoxamine 5′-phosphate>pyridoxamine>pyridoxal≥pyridoxine. The first three compounds showed higher antioxidant activity than vitamin C did. Pyridoxal 5′-phosphate prevented the reduction of the glutathione content in the MSB-treated cells and, in turn, suppressed the increases in peroxide and thiobarbituric acid reactive substances in the yeast cells and increased the viability of the yeast cells under oxidative stress. The antioxidant function of pyridoxal 5′-phosphate was not dependent on the phosphorelay pathway, which finally triggers the expression of the catalase gene.
Keywords: Vitamin B6; Menadione; Fission yeast cell; Glutathione; Reactive oxygen species; Pyridoxal 5′-phosphate;

The accumulation of extracellular matrix components such as proteoglycans is a hallmark of an atherosclerotic lesion. A large heparan sulfate proteoglycan, perlecan, dramatically increases in the advanced lesion, and vascular smooth muscle cells are the cell type responsible for the accumulation. In this study, we investigated the effects of thrombin on the proteoglycan synthesis in cultured human coronary smooth muscle cells to determine the interrelationship between the accumulation of proteoglycans and the procoagulant state of blood in atherosclerosis. The cells were metabolically labeled with [35S]sulfate or 35S-labeled amino acids in the presence of thrombin, and the labeled proteoglycans were characterized by Sepharose CL-4B molecular sieve chromatography and DEAE-Sephacel ion-exchange chromatography. The glycosaminoglycan M r and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M r was determined by SDS-polyacrylamide gel electrophoresis before and after digestion with chondroitinase ABC or papain. The results indicate that thrombin increases the cell layer-associated heparan sulfate proteoglycan with a core protein size of ∼400 kDa without any change in the length of the glycosaminoglycan chains when the cell density is high. The heparan sulfate proteoglycan was identified as perlecan by Western blot analysis. In addition, quantitative reverse transcription-polymerase chain reaction showed that thrombin elevated the steady-state level of perlecan mRNA but not that of versican, decorin, and syndecan-1 mRNAs, although that of biglycan mRNA was moderately elevated. Furthermore, the percentage of disaccharide units that compose perlecan heparan sulfate chains remained unaffected by thrombin. Therefore, it is suggested that thrombin induces the perlecan core protein synthesis without influencing the formation of the heparan sulfate chains in human coronary smooth muscle cells at a high cell density. The regulation of proteoglycan synthesis by thrombin may be involved in the accumulation of perlecan in advanced lesions of atherosclerosis.
Keywords: Atherosclerosis; Extracellular matrix; Perlecan; Proteoglycan; Thrombin; Vascular;

The aim of the present study was to evaluate the antioxidant activity of agaro-oligosaccharides with different degrees of polymerizations (DPs) and establish a relationship between the activity and DPs. The attenuate effect of oligosaccharides on 1,1-diphenyl-2-picryl-hydrazyl (DPPH) was initially assessed, and the result indicated that agarohexaose showed the highest scavenging DPPH capability (IC50=1.85 mg/ml). Following that, the intracellular antioxidant ability of agaro-oligosacharides was investigated by using the dichlorofluorescein (DCF) assay in human liver cell L-02 system. Different levels of antioxidant activities of agaro-oligosaccharides with various DPs were observed, and their scavenging reactive oxygen species (ROS) capability was associated with the improvement of the cell viability. In these oligosaccharides, agarohexaose possessed the highest scavenging capability, which could reduce 50% of oxidants generated by H2O2 at 1 mg/ml. Furthermore, the antioxidant effect of agarohexaose on the indirect oxidation of cells induced by antimycin A (AA) was also tested. The results showed that agarohexaose could scavenge ROS generated by electron leakage and protect cells against apoptosis induced by ROS. It is concluded that agaro-oligosaccharides are generally considered as novel antioxidants which could protect cell damage caused by reactive oxygen species, especially agarohexaose exhibiting most desirable effects.
Keywords: Antioxidant; Agaro-oligosaccharide; Degree of polysaccharide; Reactive oxygen species; Liver cell; Antimycin A;