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BBA - General Subjects (v.1721, #1-3)

Editorial Board (pp. ii).

Structural characterization of human liver heparan sulfate by Preeyanat Vongchan; Mohamad Warda; Hidenao Toyoda; Toshihiko Toida; Rory M. Marks; Robert J. Linhardt (pp. 1-8).

The isolation, purification and structural characterization of human liver heparan sulfate are described.¹H-NMR spectroscopy demonstrates the purity of this glycosaminoglycan (GAG) and two-dimensional¹H-NMR confirmed that it was heparan sulfate. Enzymatic depolymerization of the isolated heparan sulfate, followed by gradient polyacrylamide gel, confirmed its heparin lyase sensitivity. The concentration of resulting unsaturated disaccharides was determined using reverse phase ion-pairing (RPIP) HPLC with post column derivatization and fluorescence detection. The results of this analysis clearly demonstrate that the isolated GAG was heparan sulfate, not heparin. Human liver heparan sulfate was similar to heparin in that it has a reduced content of unsulfated disaccharide and an elevated average sulfation level. The antithrombin-mediated anti-factor Xa activity of human liver heparan sulfate, however, was much lower than porcine intestinal (pharmaceutical) heparin but was comparable to standard porcine intestinal heparan sulfate. Moreover, human liver heparan sulfate shows higher degree of sulfation than heparan sulfate isolated from porcine liver or from the human hepatoma Hep 2G cell line.

Keywords: Heparan sulfate; Human liver; Structure; Glycosaminoglycan; Proteoglycan

Characterization of NTPDase (NTPDase1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5) activity in human lymphocytes by Daniela B.R. Leal; Cristiane A. Streher; Tiago N. Neu; F�bio P. Bittencourt; Cl�udio A.M. Leal; Jos� E.P. da Silva; Vera M. Morsch; Maria R.C. Schetinger (pp. 9-15).

Human lymphocytes contain NTPDase (NTPDase-1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5), a cation-dependent enzyme that hydrolyzes ATP and ADP and also other di- and triphosphate nucleosides, acting at an optimum pH of 8.0. A significant inhibition of ATP and ADP hydrolysis ( P<0.05) was observed in the presence of 20 mM sodium azide. NTPDase inhibitors, 20 mM sodium fluoride, 0.2 mM trifluoperazine and 0.3 mM suramin, significantly decreased ATP and ADP hydrolysis ( P<0.05) and ADP hydrolysis was only inhibited by 0.5 mM orthovanadate ( P<0.05). ATP and ADP hydrolysis was not inhibited in the presence of 0.01 mM Ap5A (P¹,P⁵-di(adenosine-5′)pentaphosphate), 0.1 mM ouabain, 1 mM levamisole, 2 μg/mL oligomycin, 0.1 mM N-ethylmaleimide (NEM), or 5 mM sodium azide. With respect to kinetic behavior, apparent K_m values of 77.6±10.2 and 106.8±21.0 μM, and V_max values of 68.9±8.1 and 99.4±8.5 (mean±S.E., n=3) nmol Pi/min/mg protein were obtained for ATP and ADP, respectively. A Chevilard plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. The presence of CD39 was determined by flow cytometry, showing a low density of 2.72±0.24% (mean±S.E.; n=30) in human peripheral lymphocytes. The study of NTPDase activity in human lymphocytes may be important to determine the immune response status against infectious agents related to ATP and ADP hydrolysis.

Keywords: NTPDase; CD39; Human lymphocyte

Phosphorylation modulates the alpha-helical structure and polymerization of a peptide from the third tau microtubule-binding repeat by Jes�s Mendieta; Miguel A. Fuertes; Rani Kunjishapatham; Ismael Santa-Mar�a; Francisco J. Moreno; Carlos Alonso; Federico Gago; Victor Mu�oz; Jes�s Avila; F�lix Hern�ndez (pp. 16-26).

Paired helical filaments (PHFs) isolated from patients with Alzheimer's disease (AD) mainly consist of the microtubule-associated protein tau in a hyperphosphorylated form. It has been found that PHFs are the first example of pathological protein aggregation associated with formation of α-helices [Biochemistry (2002) 41, 7150–5]. In an effort to investigate the interplay between phosphorylation and the putative role of short regions of alpha-helix in the polymerization of tau, we have focused on the region of tau encompassing residues 317 to 335. This region is able to form protein fibrils in vitro and has two serines that are often found phosphorylated in PHFs. Using trifluoroethanol as an indicator of the alpha-helix, we find that the stability of the alpha-helix conformation is enhanced by phosphorylation. Circular dichroism data show that the phosphorylated peptide in water presents a content in alpha-helix similar to the unphosphorylated peptide at 40% of trifluoroethanol. Phosphorylation also stimulates the effect of juglone in promoting the in vitro polymerization. Furthermore, Fourier transformed infrared spectroscopy of samples of phosphorylated peptide polymerized with juglone renders a spectrum with maxima at ∼1665 and ∼1675 cm⁻¹, which are suggestive of a mixture of turns and alpha-helix conformations. Our results provide a direct mechanistic connection between phosphorylation and polymerization in tau. The connection between phosphorylation and polymerization appears to involve formation of alpha-helix structure.

Keywords: Phosphorylation; Tau; Alzheimer's disease; Circular dichroism; Molecular dynamic; Fourier transformed infrared spectroscopy

Biosynthesis of lysine in plants: evidence for a variant of the known bacterial pathways by Andre' O. Hudson; Christine Bless; Polliana Macedo; Siba P. Chatterjee; Bijay K. Singh; Charles Gilvarg; Thomas Leustek (pp. 27-36).

With the aim of elucidating how plants synthesize lysine, extracts prepared from corn, tobacco, Chlamydomonas and soybean were tested and found to lack detectable amounts of N-α-acyl-l,l-diaminopimelate deacylase or N-succinyl-α-amino-ε-ketopimelate-glutamate aminotransaminase, two key enzymes in the central part of the bacterial pathway for lysine biosynthesis. Corn extracts missing two key enzymes still carried out the overall synthesis of lysine when provided with dihydrodipicolinate. An analysis of available plant DNA sequences was performed to test the veracity of the negative biochemical findings. Orthologs of dihydrodipicolinate reductase and diaminopimelate epimerase (enzymes on each side of the central pathway) were readily found in the Arabidopsis thaliana genome. Orthologs of the known enzymes needed to convert tetrahydrodipicolinate to diaminopimelic acid (DAP) were not detected in Arabidopsis or in the plant DNA sequence databases. The biochemical and reinforcing bioinformatics results provide evidence that plants may use a novel variant of the bacterial pathways for lysine biosynthesis.

Keywords: Plant; Arabidopsis thaliana; Amino acid metabolism; Lysine; Diaminopimelate; Dihydrodipicolinate

Fructose metabolizing enzymes from mouse liver: influence of age and caloric restriction by Kevork Hagopian; Jon J. Ramsey; Richard Weindruch (pp. 37-43).

The influence of caloric restriction (CR) on the activities of liver fructose metabolizing enzymes and metabolite levels were studied in young (3 months) and old (30 months) mice. Fructokinase activity was increased ( P<0.05) in both young and old CR mice when compared to controls while triokinase activity was increased ( P<0.05) only in old CR versus control mice. Aldolase was not altered by CR in either old or young mice. No age-related differences in activities were observed in controls although a trend towards an increase was observed for triokinase, while significant age-related increases were observed for fructokinase and triokinase, but not aldolase, in CR mice. Both young and old mice on CR showed significant decreases in fructose and fructose-1-phosphate, however, no age-related changes in metabolite levels were observed for either control or CR mice. A fructose-1-phosphate kinase activity was also measured and found to be unchanged in both young and old mice on CR, but the activity was significantly lower in the old mice compared with young. We show here that the enzymes involved in fructose metabolism are influenced by CR and that this could contribute to alterations in gluconeogenesis and glycolysis observed with CR.

Keywords: Abbreviations; CR; caloric restriction; F-1-P; fructose-1-phosphate; F-1,6-BP; fructose-1,6-bisphosphate; DHAP; dihydroxyacetone phosphate; GAP; glyceraldehyde-3-phosphate; 1-PFK; 1-phosphofructokinase; FK; fructokinase; TK; triokinase; PFK-1; phosphofructokinase-1Aging; Aldolase; Calorie restriction; Fructokinase; Fructose-1-phosphate; Triokinase

Glycosaminoglycan destabilization of DNA–chitosan polyplexes for gene delivery depends on chitosan chain length and GAG properties by Signe Danielsen; Sabina Strand; Catharina de Lange Davies; Bj�rn T. Stokke (pp. 44-54).

Chitosan-based gene delivery systems are promising candidates for non-viral gene therapy. A wide range of chitosans has been studied to optimize the properties of the DNA–chitosan complexes to yield high transfection efficiencies. An important parameter to control is the polyplex stability to allow transport towards the cells, subsequent internalization and release of DNA intracellularly. The stability of the DNA–chitosan complexes was here studied after exposure to heparin and hyaluronic acid (HA) using atomic force microscopy (AFM) and ethidium bromide (EtBr) fluorescence assay. To study the effect of polycation chain length on the polyplex stability, chitosans with a degree of polymerization (DP) varying from ∼10 to ∼1000 were employed for DNA compaction. Whereas HA was unable to dissociate the complexes, the degree of dissociation caused by heparin depended on both the chitosan chain length and the amount of chitosan used for complexation. When increasing the chitosan concentration, larger heparin concentrations were required for polyplex dissociation. Furthermore, increasing the chitosan chain length yielded more stable complexes. Varying the chitosan chain length thus provides a tool for controlling the ability of the polyplex to deliver therapeutic gene vectors to cells.

Keywords: Chitosan; DNA; Polyplex; Stability; AFM; Fluorescence

Induction of apoptosis by electrotransfer of positively charged proteins as Cytochrome C and Histone H1 into cells by I. Tsoneva; B. Nikolova; M. Georgieva; M. Guenova; T. Tomov; M.-P. Rols; M.R. Berger (pp. 55-64).

Cytochrome C (Cyt. C) is a mitochondrial protein inducing apoptosis when it is accumulated in the cytosol by a currently unknown mechanism, but regulated by the bcl-2 family of proteins. The linker Histone H1 is another basic protein with highly conservative structure, composition, and equal molecular weight, not changed during the evolution.An attempt was made to understand better the apoptotic processes by electroloading of leukemic cells, such as K562, HL-60, and SKW3, and human lymphocytes with positively charged proteins, such as Cyt. C, Histone H1, and methylated BSA albumin (mBSA). The triggering apoptotic processes followed by MTT test, FACS analysis, and DNA fragmentation after the electrotransfer of these proteins into the cells were observed.Histone H1 and mBSA induce the release of Cyt. C from rat liver mitochondria. Cytochrome C release was higher when mitochondria were in “high-energy�? state. It is supposed that release of Cyt. C from mitochondria is due to the mechanical rupture of the outer mitochondrial membrane, rich in negatively charged groups, predominately due to cardiolipin. The reason for the morphological rupture of the outer mitochondial membrane could be the rigidification and segregation of the membrane and the destroyed membrane asymmetries of both monolayers in the presence of positively charged proteins at higher linear charges such as Histone H1. We suggested that Histone H1, at a given moment of activated signaling for apoptosis, could be not transported to the nucleus and could lead to the release of Cyt. C from the mitochondria in the cytoplasm. It is temping to speculate that Histone H1 has other physiological extranuclear functions involved in apoptosis.

Keywords: Apoptosis; Cytochrome; C; Histone H1; methylated BSA (mBSA); Electroporation; Mitochondria

Novel nonmatrix-metalloproteinase-mediated collagen degradation by F. Song; L.J. Windsor (pp. 65-72).

Matrix metalloproteinases (MMPs) and their inhibitors have long been believed to play a major role in the collagen loss seen in destructive temporomandibular joint (TMJ) disorders. This project was originally designed to examine the expression of MMPs and the tissue inhibitors of MMPs (TIMPs) by diseased human TMJ synovial fibroblasts and to determine their ability to degrade Type I collagen. Reverse transcriptase-PCR indicated that these TMJ synovial fibroblasts expressed mRNA for multiple MMPs and TIMPs. The collagen degradation assay showed that these TMJ synovial fibroblasts at passage 3 to 8 were capable of digesting the collagen underneath them on collagen-coated plates. This degradation was inhibited by GM6001, a synthetic MMP inhibitor. During passage 8 to 13, these TMJ fibroblasts were able to digest all the collagen in the wells. This degradation was completely inhibited by combining GM6001 and soybean trypsin inhibitor (STI), a serine proteinase inhibitor. The collagen cleavage activity of collected conditioned media was dramatically inhibited by STI but not by 1,10-phenanthroline, an MMP inhibitor. The data suggest that these TMJ cells utilize a MMP-dependent pathway and a novel MMP-independent pathway to digest Type I collagen.

Keywords: Destructive temporomandibular joint disorder; Collagen degradation; Matrix metalloproteinase; Fibroblast; Serine proteinase

cDNA microarray analysis of lactoferrin expression in non-neoplastic human hepatocyte PH5CH8 cells by Takahiko Tamura; Akito Nozaki; Ken-ichi Abe; Hiromichi Dansako; Kazuhito Naka; Masanori Ikeda; Katsuaki Tanaka; Nobuyuki Kato (pp. 73-80).

Lactoferrin (LF), a milk protein belonging to the iron transporter transferrin family, is known as a primary defense protein against pathogenic microorganisms. Previously, we found that bovine and human LFs prevented hepatitis C virus infection in cultured human hepatocytes by a direct interaction with the virus. Since LF is proposed to have transcriptional regulatory activity in addition to its antimicrobial function, we sought to identify the target genes that these two types of LF have in common. To this end, we were the first to perform microarray analysis (9970 genes) using human hepatocytes that expressed bovine or human LF by retrovirus-mediated gene transfer. In the results, LF could give a variety of expression profiles in the human hepatocytes, and showed that 9 and 19 genes were commonly up-regulated (more than 2.0-fold) and down-regulated (less than 0.50-fold), respectively, in both bovine and human LF-expressing cells compared with control cells. Among these genes, we found that γ-aminobutyric acid (GABA)-B receptor 2 was transcriptionally down-regulated by bovine and human LFs, but not by human transferrin. Furthermore, we obtained the suggestive result that LF may modulate the level of intracellular cAMP. This modulation is one of the cellular responses that the GABA-B receptor modifies. This is the first report of microarray analysis applied to search inclusively for the target genes of LF.

Keywords: Lactoferrin; PH5CH8; Microarray; GABA-B receptor; cAMP; Hepatitis C virus

The extractability of extracellular matrix components as a marker of cartilage remodeling in laryngeal squamous cell carcinoma by Spyros S. Skandalis; Dimitrios A. Theocharis; Nickoletta Papageorgakopoulou; Demitrios H. Vynios (pp. 81-88).

Sequential extraction was applied to investigate the proteoglycan (PG) organization in healthy laryngeal cartilage (HLC) and laryngeal cartilage squamous cell carcinoma (LCSCC). Highly stable aggrecan aggregates, extracted from both HLC and LCSCC with strong dissociative reagents, i.e., 4 M guanidine HCl (GdnHCl), represented 53% and 7%, respectively, of total extracted macromolecules. Less stable complexes/aggregates, extracted with mild dissociative reagents (1 and 2 M GdnHCl), represented 40% and 61% of total extracted PGs from healthy and cancerous cartilage, respectively. Interestingly, a relative high proportion (32%) of uronic acid (UA)-containing macromolecules were removed from the cancerous cartilage using associative extracting solutions (PBS and 0.5 M GdnHCl), which obviously represented molecules freely extractable from the tissue. In contrast, the corresponding proportion in HLC was impressively low (about 7%). The major proportion of these molecules was chondroitin sulfate-containing PGs (CSPGs), which identified mainly as aggrecan. Differential digestion of the sequential extracts with chondroitinase ABC and chondroitinase AC II demonstrated the presence of dermatan sulfate-containing PGs (DSPGs) in both HLC and LCSCC, being mainly present in the 1 M GdnHCl extract, and identified as decorin. All cancerous extracts were found to be rich in 4-sulfated disaccharides, mostly participating in DS structures. In conclusion, the applied procedure permitted the elucidation of the changes in the cartilage status, regarding the stability and identity of its proteoglycan aggregates/complexes, in both HLC and LCSCC.

Keywords: Aggrecan; Decorin; Chondroitin/dermatan sulfate; Degradation; Extractability

Peptides and hydrolysates from casein and soy protein modulate the release of vasoactive substances from human aortic endothelial cells by R. Ringseis; B. Matthes; V. Lehmann; K. Becker; R. Sch�ps; R. Ulbrich-Hofmann; K. Eder (pp. 89-97).

Food proteins were shown to affect atherogenic risk factors, which is supposed to be related to specific peptide sequences encrypted within their primary sequence. The aim of this study was to evaluate the effects of peptides and hydrolysates from two food proteins, casein and soy protein, on endothelial cell functions (cell proliferation and release of vasoactive substances). Cell proliferation was not influenced by dipeptides and most of the tripeptides, whereas several total hydrolysates from casein and soy protein inhibited cell proliferation at higher concentrations (>0.25 mg/mL; P<0.05). The release of one or more of the vasoactive substances, thromboxan B₂ (stable marker of thromboxan A₂), 6-keto-prostaglandin F_1α (stable marker of prostaglandin I₂), endothelin-1, and nitric oxide, was significantly influenced by the incubation with various peptides compared with control cells ( P<0.05). Various hydrolysate fractions from casein and soy protein influenced the release of 6-keto-prostaglandin F_1α and nitric oxide ( P<0.05) but did not influence the release of thromboxan B₂ and endothelin-1. In conclusion, the present study demonstrates that peptides and hydrolysate fractions from casein and soy protein influence endothelial cell function as evidenced by the modulation of endothelial cell proliferation and alterations in the release of vasoactive substances.

Keywords: Abbreviations; ACE; angiotensin-I-converting enzyme; BSA; bovine serum albumin; EDTA; ethylenediaminetetraacetate; C; chymotrypsin; E; elastase; ET-1; endothelin-1; HBSS; Hank's buffered salt solution; HEPES-BSS; HEPES buffered salt solution; mTOR; mammalian Target of Rapamycin; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NO; nitric oxide; P; pepsin; PGI; ₂; prostaglandin I; ₂; T; trypsin; TXA; ₂; thromboxan A; ₂; TXB; ₂; thromboxan B; ₂; TNFα; tumor necrosis factor-α; TNS; trypsin neutralizing solution; VEGF; vascular endothelial growth factor; 6-keto-PGF; _1α; 6-keto-prostaglandin; _1αPeptide; Hydrolysate; Endothelial cell; Casein; Soy protein

Low tumor cell density environment yields survival advantage of tumor cells exposed to MTX in vitro by Josep M. de Anta; Francisco X. Real; Xavier Mayol (pp. 98-106).

Stable resistance to methotrexate has been well characterized after prolonged treatment of the HT-29 colon cancer cell line, but the mechanism of cell survival at the early stages of the drug resistance process still remains unclear. Here, we demonstrate that human cancer cells in vitro are sensitive to methotrexate only above a critical cell culture density, which specifically coincides with their ability to deplete the extracellular nucleosides from a fully supplemented culture medium. At lower cell densities, extracellular nucleosides remain intact and allow salvage nucleotide synthesis that renders cells insensitive to the drug. Consistently, medium conditioned by cells seeded at standard cell densities sensitizes low cell density cultures. Extracellular nucleosides are the determinants of sensitivity because the latter effect can be mimicked with the use of inhibitors of nucleoside cellular import and reversed by supplying exogenous thymidine and hypoxanthine. Interestingly, treatment at a sensitizing cell density does not preclude the survival of less than 1% of the cells—which have no intrinsic resistance—owing to the inability of the dying cell population to condition the culture medium; this population thus survives indefinitely to continuous treatment by keeping adapted to a low cell number. This cell density-dependent adaptive process accounts for the initial steps of in vitro resistance to methotrexate (MTX) and provides a novel mechanistic insight into the cell population dynamics of cell survival and cell death during drug treatment.

Keywords: Abbreviations; DHFR; dihydrofolate reductase gene; FBS; fetal bovine serum; IC50; 50% inhibitory drug concentration; LCD; low cell density; MTX; methotrexate; NBMPR; nitrobenzyl-mercaptopurine ribonucleoside; DP; dipyridamole; SCD; standard cell densityDrug resistance; Neoplasm [G12.392.395]; Tumor cell; Cultured [A11.251.210.770]; Cell survival [G04.335.316]; Nucleoside [D13.570]; Neoplasm; Residual [C04.697.700]

Detection of glycosyltransferases in the golden hamster ( Mesocricetus auratus) oviduct and evidence for the regulation of O-glycan biosynthesis during the estrous cycle by Deborah S. McBride; Inka Brockhausen; Frederick W.K. Kan (pp. 107-115).

Recently, we provided evidence that the glycosylation of hamster oviductin, a member of the mucin family of glycoproteins, is regulated during the estrous cycle. In order to further elucidate the glycosylation process of oviductal glycoproteins, we identified biosynthetic pathways involved in the assembly of mucin-type O-linked oligosaccharide ( O-glycan) chains in the hamster oviduct. Our results demonstrated that the hamster oviduct has high activities of glycosyltransferases that synthesize O-glycans with core 1, 2, 3 and 4 structures as well as elongated structures. Oviduct therefore represents a typical mucin-secreting tissue. Our results also showed that specific glycosyltransferase activities are regulated during the estrous cycle. Mucin-type core 2 β6-GlcNAc-transferase (C2GnT2) is responsible for synthesizing core 2 and core 4 structures in the oviduct. Specific assays for C2GnT2 revealed a cyclical pattern throughout the estrous cycle with high activity at the stages of proestrus and estrus and low activity at diestrus 1. Using semiquantitative RT-PCR, the mRNA levels for C2GnT2 in the estrous cycle stages could be correlated with the enzyme activities. An increase in glycosyltransferase activity in the hamster oviduct at the time of ovulation suggests that glycosylation of oviductal glycoproteins may be necessary for these proteins to exert their functions during the process of fertilization.

Keywords: Abbreviations; Ac; acetyl amide; Bpa; 4-benzoyl-; l; -phenylalanine; Bn; benzyl; HPLC; high-performance liquid chromatography; HVE; high-voltage electrophoresis; PBS; phosphate-buffered saline; PNP; para; -nitrophenol; RT-PCR; reverse transcription polymerase chain reactionGlycosyltransferase; Oviduct; Estrous cycle; Steroid hormone; Golden hamster

An extracellular endodeoxyribonuclease from Streptomyces aureofaciens by Zuzana Brn�kov�; Andrej God�ny; Jozef Timko (pp. 116-123).

Several extracellular DNases were detected after cultivation of Streptomyces aureofaciens B96 under submerged conditions. These DNases are nutritionally regulated and high content of amino acid nitrogen in cultivation medium repress their production. By varying cultivation conditions, there remained only two extracellular nuclease activities. The major one, extracellular endodeoxyribonuclease SaD I, was purified to homogeneity by ammonium sulfate precipitation, adsorption on Spheron, chromatography on Superose-12P followed by FPLC on MonoQ and final purification on HiTrapQ. The molecular weight of the purified SaD I determined by SDS-PAGE was 31 kDa. The DNase hydrolyses endonucleolytically both double-stranded and single-stranded circular and linear DNA. It does not cleave RNA and does not exhibit phosphodiesterase nor phosphomonoesterase activity. It requires a divalent cation (Zn²⁺, Co²⁺, Mn²⁺, Mg²⁺) and its activity optimum is at neutral pH (pH 7.2). The optimal temperature for DNA cleavage was 40 �C. Activity was strongly inhibited in the presence of phosphate, Hg²⁺, chelating agents or iodoacetate, but it was stimulated by addition of dimethyl sulphoxide.

Keywords: Streptomyces aureofaciens; nuclease; SaD I; Endodeoxyribonuclease; Extracellular nuclease; Purification

Inhibition of firefly luciferase by alkane analogues by K� Takehara; Hiroshi Kamaya; Issaku Ueda (pp. 124-129).

We reported that anesthetics increased the partial molal volume of firefly luciferase (FFL), while long-chain fatty acids (LCFA) decreased it. The present study measured the actions of dodecanol (neutral), dodecanoic acid (negatively charged), and dodecylamine (positively charged) hydrophobic molecules on FFL. The interaction modes are measured by (1) ATP-induced bioluminescence of FFL and (2) fluorescence of 2-( p-toluidino)naphthalene-6-sulfonate (TNS). TNS fluoresces brightly in hydrophobic media. It competes with the substrate luciferin on the FFL binding. From the Scatchard plot of TNS titration, the maximum binding number of TNS was 0.83, and its binding constant was 8.27×10⁵ M⁻¹. Job's plot also showed that the binding number is 0.89. From the TNS titration of FFL, the binding constant was estimated to be 8.8×10⁵ M⁻¹. Dodecanoic acid quenched the TNS fluorescence entirely. Dodecanol quenched about 25% of the fluorescence, whereas dodecylamine increased it. By comparing the fluorescence of TNS and bioluminescence of FFL, the binding modes and the inhibition mechanisms of these dodecane analogues are classified in three different modes: competitive (dodecanoic acid), noncompetitive (dodecylamine), and mixed (dodecanol).

Keywords: Anesthesia mechanism; Anesthesia antagonist; Thermal transition of protein; Firefly luciferase

Boundary-element calculations for dielectric behavior of doublet-shaped cells by Katsuhisa Sekine; Yoko Watanabe; Saori Hara; Koji Asami (pp. 130-138).

In order to simulate dielectric relaxation spectra (DRS) of budding yeast cells ( Saccharomyces cerevisiae) in suspension, the complex polarization factor (Clausius–Mossotti factor) β for a single cell and the complex permittivity of a cell suspension ε_sus* were calculated with a doublet-shaped model (model RD), in which two spheres were connected with a part of a ring torus, using the boundary element method. The β values were represented by a diagonal tensor consisting of components β_z parallel to the rotation axis ( z axis) and β_h in a plane ( h plane) perpendicular to the axis. The ε_sus* values were calculated from the complex permittivity of the suspending medium ε_a* and the components of β. The calculation was compared with that of a conventional prolate spheroid model (model CP). It was found that model CP could be used as a first approximation to model RD. However, differences existed in β_z between models RD and CP; β_z showed three relaxation terms in the case of model RD in contrast with two terms in model CP. Narrowing the junction between the two spheres in model RD markedly decreased the characteristic frequency of one of the relaxation terms in β_z. This suggests that the structure of the junction can be estimated from DRS. Effects of the shape change from model RD to a two-sphere model (model RD without the junction) were also examined. The behavior of β_z in the two-sphere model, the relaxation intensity of which was much lower than model RD, was quite similar to that in a single-sphere model. These simulations were consistent with the experimental observations of the dielectric behavior of the yeast cells during cell cycle progression.

Keywords: Clausius–Mossotti factor; Complex permittivity; Dielectric relaxation; Interfacial polarization; Saccharomyces cerevisiae; Simulation

Do clustered β-propeller domains within the N-terminus of LRP1 play a functional role? by Fengcheng Sun; Rita Kohen Avramoglu; Gerard Vassiliou; Robert J. Brown; Kerry W.S. Ko; Ruth McPherson; Zemin Yao (pp. 139-151).

The six β-propellers located within the N-terminus of low density lipoprotein receptor-related protein 1 (LRP1) are arranged in two clusters that contain two and four β-propellers, respectively. Working with LRP1 deletion mutants, we found that randomly removing large segments of amino acid sequences did not affect the intracellular trafficking of LRP1 as long as the clustered β-propeller domains were retained. However, deletion mutants with crippled β-propeller clusters invariably exhibited retarded exit from the endoplasmic reticulum (ER). To determine potential functions of the clustered β-propellers, we generated a series of deletion mutants in which the β-propellers were systematically removed from the C-terminal end of the second cluster. The resulting minireceptors, designated LRPβ1–6, β1–5, β1–4, β1–3, and β1–2 containing decreasing numbers of the β-propellers, were stably expressed in LRP1-null CHO cells. Binding/degradation assays with receptor-associated protein or α₂-macroglobulin showed that removing one or more β-propellers had little effect on binding or degradation of these ligands. However, minireceptors containing odd number of β-propellers (i.e., LRPβ1–3 and β1–5) showed prolonged retention within the ER and remained endoglycosidase H-sensitive, whereas minireceptors containing even number of β-propellers (i.e., LRPβ1–2, β1–4 and β1–6) exited ER at variable rates. Cell surface biotinylation experiments showed that LRPβ1–3 was absent from the cell surface. Prolonged retention of LRPβ1–3 within the ER was accompanied by increased association with molecular chaperone Grp78/Bip. These results suggest that the clustered β-propellers may play a role in folding and intracellular trafficking of LRP1.

Keywords: Abbreviations; LRP1; low density lipoprotein receptor-related protein 1; LDLR; low density lipoprotein receptor; ER; endoplasmic reticulum; EGF; epidermal growth factor; RAP; receptor-associated protein; CHO; Chinese hamster ovary; endo H; endoglycosidase H; PNGaseF; peptide:; N; -glycosidase F; FBS; fetal bovine serum; DMEM; Dulbecco's modified Eagle medium; PBS; phosphate-buffered saline; PAGE; polyacrylamide gel electrophoresis; α; ₂; M; α; ₂; -macroglobulin; HBSS; Hanks balanced salt solution; BSA; bovine serum albumin; TCA; trichloroacetic acidLRP minireceptor; β-propeller; CHO LRP1-null cell; Trafficking

Lectin KM⁺-induced neutrophil haptotaxis involves binding to laminin by Luciane Ganiko; Ant�nio R. Martins; Edna Freym�ller; Renato A. Mortara; Maria-Cristina Roque-Barreira (pp. 152-163).

The lectin KM⁺ from Artocarpus integrifolia, also known as artocarpin, induces neutrophil migration by haptotaxis. The interactions of KM⁺ with both the extracellular matrix (ECM) and neutrophils depend on the lectin ability to recognize mannose-containing glycans. Here, we report the binding of KM⁺ to laminin and demonstrate that this interaction potentiates the KM⁺-induced neutrophil migration. Labeling of lung tissue by KM⁺ located its ligands on the endothelial cells, in the basement membrane, in the alveolus, and in the interstitial connective tissue. Such labeling was inhibited by 400 mMd-mannose, 10 mM Manα1-3[Manα1-6]Man or 10 μM peroxidase (a glycoprotein-containing mannosyl heptasaccharide). Laminin is a tissue ligand for KM⁺, since both KM⁺ and anti-laminin antibodies not only reacted with the same high molecular mass components of a lung extract, but also determined colocalized labeling in basement membranes of the lung tissue. The relevance of the KM⁺-laminin interaction to the KM⁺ property of inducing neutrophil migration was evaluated. The inability of low concentrations of soluble KM⁺ to induce human neutrophil migration was reversed by coating the microchamber filter with laminin. So, the interaction of KM⁺ with laminin promotes the formation of a substrate-bound KM⁺ gradient that is able to induce neutrophil haptotaxis.

Keywords: Abbreviations; Ab; antibody; ANOVA; analysis of variance; BSA; bovine serum albumin; CRD; carbohydrate recognition domain; DIC; differential interference contrast; ECM; extracellular matrix; EDTA; ethylenediaminetetraacetic acid; EHS; Elgelbreth–Holm–Swarm; fMLP; formyl-methionyl-leucyl-phenylalanine; ICAM; intercellular adhesion molecule; Ig; immunoglobulin; IL-8; interleukin-8; HRP; horseradish peroxidase; MCP-1; monocyte chemoattractant protein-1; 2-ME; 2-mercaptoethanol; MIP-1α; macrophage inflammatory protein-1α; MNCF; macrophage-derived neutrophil chemotactic factor; PAGE; polyacrylamide gel electrophoresis; PBS; phosphate-buffered saline; PECAM-1; platelet–endothelial cell adhesion molecule-1; PMSF; phenylmethylsulfonyl fluoride; PVP; polyvinylpyrrolidone; S.E.; standard error of the mean; SDS; sodium dodecyl sulfateLectin; Artocarpus integrifolia; Neutrophil haptotaxis; Extracellular matrix; Laminin; Haptotactic gradient

Flavonoid–serum albumin complexation: determination of binding constants and binding sites by fluorescence spectroscopy by Claire Dufour; Olivier Dangles (pp. 164-173).

After a meal rich in plant products, dietary flavonols can be detected in plasma as serum albumin-bound conjugates. Flavonol–albumin binding is expected to modulate the bioavailability of flavonols. In this work, the binding of structurally different flavonoids to human and bovine serum albumins is investigated by fluorescence spectroscopy using three methods: the quenching of the albumin fluorescence, the enhancement of the flavonoid fluorescence, the quenching of the fluorescence of the quercetin–albumin complex by a second flavonoid. The latter method is extended to probes whose high-affinity binding sites are known to be located in one of the two major subdomains (warfarin and dansyl-l-asparagine for subdomain IIA, ibuprofen and diazepam for subdomain IIIA). Overall, flavonoids display moderate affinities for albumins (binding constants in the range 1–15×10⁴ M⁻¹), flavones and flavonols being most tightly bound. Glycosidation and sulfation could lower the affinity to albumin by one order of magnitude depending on the conjugation site. Despite multiple binding of both quercetin and site probes, it can be proposed that the binding of flavonols primarily takes place in subdomain IIA. Significant differences in affinity and binding location are observed for the highly homologous HSA and BSA.

Keywords: Abbreviations; HSA; human serum albumin; BSA; bovine serum albumin; DNSA; dansyl-; l; -asparagine; HAS; high-affinity site; LAS; low-affinity siteSerum albumin; Flavonoid; Fluorescence; Binding site; Binding constant

Evaluation of the antioxidant activity of flavonoids by “ferric reducing antioxidant power�? assay and cyclic voltammetry by Omidreza Firuzi; Antonio Lacanna; Rita Petrucci; Giancarlo Marrosu; Luciano Saso (pp. 174-184).

Flavonoids, naturally occurring phenolic compounds, have recently been studied extensively for their antioxidant properties. The structure–antioxidant activity relationships (SAR) of flavonoids have been evaluated against different free radicals, but “ferric reducing antioxidant power�? (FRAP) assay, which determines directly the reducing capacity of a compound, has not been used for this purpose. In this study, the antioxidant activities of 18 structurally different flavonoids were evaluated by FRAP assay modified to be used in 96-well microplates. Furthermore, their oxidation potentials were also measured, which were in the range of +0.3 V (myricetin) to +1.2 V (5-hydroxy flavone) and were in good agreement with FRAP assay results. Quercetin, fisetin and myricetin had the lowest oxidation potentials and appeared the most active compounds in FRAP assay and were 3.02, 2.52 and 2.28 times more active than Trolox, respectively. Indications were found that the o-dihydroxy structure in the B ring and the 3-hydroxy group and 2,3-double bond in the C ring give the highest contribution to the antioxidant activity.

Keywords: Abbreviations; E; _ap; anodic potential; E; _cp; cathodic potential; FRAP; ferric reducing antioxidant power; SAR; structure–activity relationship; TEAC; Trolox equivalent antioxidant capacity; TPTZ; 2,4,6-Tris(2-pyridyl)-; s; -triazineAntioxidant; Flavonoid; FRAP; Cyclic voltammetry

Regulation of acetylated tubulin/Na⁺,K⁺-ATPase interaction byl-glutamate in non-neural cells: involvement of microtubules by C�sar H. Casale; Gabriela Previtali; Juan J. Serafino; Carlos A. Arce; H�ctor S. Barra (pp. 185-192).

A subpopulation of membrane tubulin consisting mainly of the acetylated isotype is associated with Na⁺,K⁺-ATPase and inhibits the enzyme activity. We found recently that treatment of cultured astrocytes withl-glutamate induces dissociation of the acetylated tubulin/Na⁺,K⁺-ATPase complex, resulting in increased enzyme activity. We now report occurrence of this phenomenon in non-neural cells. As in the case of astrocytes, the effect ofl-glutamate is mediated by its transporters and not by specific receptors. In COS cells, the effect ofl-glutamate was reversed by its elimination from culture medium, provided thatd-glucose was present. The effect ofl-glutamate was not observed when Na⁺ was replaced by K⁺ in the incubation medium. The ionophore monensin, in the presence of Na⁺, had the same effect asl-glutamate. Treatment of cells with taxol prevented the dissociating effect ofl-glutamate or monensin. Nocodazole treatment of intact cells or isolated membranes dissociated the acetylated tubulin/Na⁺,K⁺-ATPase complex. The dissociating effect of nocodazol does not require Na⁺. These results indicate a close functional relationship among Na⁺,K⁺-ATPase, microtubules, andl-glutamate transporters, and a possible role in cell signaling pathways.

Keywords: Acetylated tubulin/Na; ⁺; ,K; ⁺; -ATPase complex; Microtubule; Non-neural cell

Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea by Patricia Lucas-Elio; Pilar Hernandez; Antonio Sanchez-Amat; Francisco Solano (pp. 193-203).

This work describes the purification and partial characterization of a novel antibacterial compound, here named marinocine, produced by Marinomonas mediterranea, a melanogenic marine bacterium with rich secondary metabolism. The antibacterial compound is a protein detected in the medium at death phase of growth. It has been purified to apparent homogeneity from the supernatants of cultures by means of ethanol precipitation followed by column chromatographies on DEAE-Sephadex and Sephacryl HR-200. The protein has an apparent molecular mass of 140–170 kDa according to gel permeation chromatography and non-denaturing SDS-PAGE, although in denaturing SDS-PAGE two mayor bands of 97 and 185 kDa appear. Marinocine is relatively heat-stable and shows a great resistance against many hydrolytic enzymes such as glycosidases, lipase, and proteases. The antibacterial range of the molecule includes Gram-positive and Gram-negative microorganisms, as well as some nosocomial isolates, Staphylococcus aureus and Pseudomonas sp., highly resistant to classical antibiotics. By contrast, marinocine did not show any effect on the eukaryotic microorganisms tested. Regarding eukaryotic CHO cells, the decrease on viability was much lower than the one observed on bacterial cells.

Keywords: Abbreviations; CFU; colony forming units; CHO; Chinese hamster ovary; DEAE; diethylaminoethyl; DHI; 5,6-dihydroxyIndole; DHN; 1,8-dihydroxynaphthalene; LB; Luria-Bertani medium; MBC; minimal bactericidal concentration; MIC; minimal inhibitory concentration; MMC; complex marine medium; MMM; minimum marine medium; PPO; polyphenol oxidasePigmentation; Antibacterial protein

Author Index (pp. 205-206).

Cumulative Contents (pp. 207-208).

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BBA - General Subjects (v.1721, #1-3)