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BBA - General Subjects (v.1760, #1)

Editorial Board (pp. ii).

Cryptotanshinone inhibits endothelin-1 expression and stimulates nitric oxide production in human vascular endothelial cells by Zhe Zhou; Sheng-Qi Wang; Yong Liu; Ai-Dong Miao (pp. 1-9).

The Chinese herb Salvia miltiorrhiza (SM) has been found to have beneficial effects on the circulatory system. In the present study, we investigated the effects of cryptotanshinone (derived from SM) on endothelin-1 (ET-1) expression in human umbilical vein endothelial cells (HUVECs). The effect of cryptotanshinone on nitric oxide (NO) in HUVECs was also examined. We found that cryptotanshinone inhibited basal and tumor necrosis factor-α (TNF-α) stimulated ET-1 secretion in a concentration-dependent manner. Cryptotanshinone also induced a concentration-dependent decrease in ET-1 mRNA expression. Cryptotanshinone increased basal and TNF-α-attenuated NO production in a dose-dependent fashion. Cryptotanshinone induced a concentration-dependent increase in endothelial nitric oxide synthase (eNOS) expression without significantly changing neuronal nitric oxide synthase (nNOS) expression in HUVECs in the presence or absence of TNF-α. NOS activities in the HUVECs were also induced by cryptotanshinone. Furthermore, decreased ET-1 expression in response to cryptotanshinone was not antagonized by the NOS inhibitor l-NAME. A gel shift assay further showed that TNF-α-induced Nuclear Factor-κB (NF-κB) activity was significantly reduced by cryptotanshinone. These data suggest that cryptotanshinone inhibits ET-1 production, at least in part, through a mechanism that involves NF-κB but not NO production.

Keywords: Cryptotanshinone; Salvia miltiorrhiza; Endothelin-1; Nitric oxide; Vascular endothelial cell

Characterization of a methionine adenosyltransferase over-expressing strain in the trypanosomatid Leishmania donovani by Yolanda P�rez-Pertejo; Rosa M. Reguera; David Ord��ez; Rafael Bala�a-Fouce (pp. 10-19).

Methionine adenosyltransferase (MAT: EC 2.5.1.6) catalyzes the synthesis of S-adenosylmethionine (AdoMet) in two sequential steps, AdoMet formation and subsequent tripolyphosphate (PPPi) cleavage, induced by AdoMet. In pursuit of a better understanding of the biological function of the enzyme, the MAT gene was cloned into vector PX63NEO to induce episomal overexpression in leishmania parasites. Neomycin-selected clones originated a strain of such overexpressing parasites that accumulated more than 3-fold AdoMet than mock-transfected cells and showed over ten times the wild type MAT activity, concurring with a significant accumulation of the MAT protein during the early logarithmic phase and MAT transcripts throughout the growth cycle. The rate of AdoMet efflux, practically nil in the control promastigotes, was exceptionally high in the MAT-overexpressing parasites, whilst growth in this strain was comparable to development in control cells, i.e., it was not affected by deleterious hypermethylation. Moreover, the modified strain was 10-fold more resistant to sinefungin, a S-adenosylmethionine-like antibiotic, than control cells. The effects of overexpression on polyamine metabolism and transport were likewise studied.

Keywords: Abbreviations; MAT; methionine adenosyltransferase; AdoMet; S-adenosylmethionine; AdoHcy; S-adenosylhomocysteine; DFMO; α-difluoromethylornithine; SUMO; Small Ubiquitin-Related Modifier; ORF; Open Reading FrameMethionine adenosyltransferase; S-adenosylmethionine; Polyamine; Leishmania donovani; Trypanosomatid

Solubilization, purification and reconstitution of Ca²⁺-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC by Amritlal Mandal; Sudip Das; Tapati Chakraborti; Pulak Kar; Biswarup Ghosh; Sajal Chakraborti (pp. 20-31).

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.

Keywords: Abbreviations; ER; endoplasmic reticulum; SR; sarcoplasmic reticulam; DHPC; 1,2-diheptanoyl-sn-phosphatidylcholine; C; ₁₂; E; ₈; poly(oxy-ethylene)8-lauryl ether; DOPC; dioleoyl-phosphatidylcholine; DTT; D,L-1,4-dithiothreitol; HBPS; Hank's buffered physiological saline; MOPS; 3-(; N; -morpholino)propanesulphonic acid; TBS; tris-buffered saline; EGTA; ethylene glycol bis(; 2; -aminoethyl ether)-; N,N,N′,N′-; tetraacetic acid; PMSF; phenylmethylsulfonyl fluoride; DFBAPTA; 5-5′ difluoro derivative of 1,2-bis(o-aminophenoxy)ethane-; N,N,N; ′,; N; ′,tetraacetic acid; CD; circular dichroism; FCCP; carbonylcyanide-; p; -trifluoromethoxyphenylhydrazoneCa; ²⁺; -ATPase; Endoplasmic reticulum; Bovine pulmonary artery smooth muscle microsome; Liposome; DHPC; C; ₁₂; E; ₈; Triton X-100; DOPC; FCCP

Increase in phosphotidylinositide-3 kinase activity by nitrotyrosylation of lysates of platelets from patients with systemic sclerosis by Thomas M. Chiang; Arnold E. Postlethwaite (pp. 32-37).

We have observed that the platelet non-integrin collagen receptor (65 kDa) and another protein ( M_r 185 kDa) are altered in the posttranslational modification by nitrotyrosylation in platelets from patients with systemic sclerosis (SSc). We reported the identification of nitrotyrosylated 65-kDa proteins in a previous study. In the present investigation, using Western blots, one- and two-dimensional gel electrophoreses and matrix assisted ionization/desorption-time of flight (MALDI-TOF) we have identified the 185-kDa protein as phosphoinositide kinase C2β (PI 3-K). There is a positive correlation between the nitrotyrosylation of PI 3-K and activity of the enzyme, i.e., the nitrotyrosylation of PI 3-K increases its enzymatic activity. In addition, the activity of PI 3-K increases in nitrotyrosylated platelet lystaes from patients with SSc compared to normal volunteer controls, suggesting that this is an alteration in the posttranslational modification of PI 3-K in platelets from patients with SSc. The increased nitrotyrosylation of PI 3-K may contribute to the impairment of platelet function in patients with SSc by increasing platelet reactivity to matrix components within the vascular walls of patients with this disease.

Keywords: Platelet; Nitrotyrosylation of protein; Systemic sclerosis; Nitric oxide synthase; Collagen; PI 3-kinase

The pH-dependent conformational transition of β-lactoglobulin modulates the binding of protoporphyrin IX by Fang Tian; Katrina Johnson; Andrea E. Lesar; Harry Moseley; James Ferguson; Ifor D.W. Samuel; Alberto Mazzini; Lorenzo Brancaleon (pp. 38-46).

We have investigated the interaction between PPIX and β-lactoglobulin (β-lg) as a function of the pH of the solution. β-lg is a small globular protein (MW ≈18 kDa) with a very well characterized structure that reveals several possible binding sites for ligands. The interaction with β-lg affects the photophysical properties of PPIX. The shift of PPIX emission maximum, excitation maximum and the increase of the fluorescence intensity is an indicator that binding between the porphyrin and β-lg occurs. The binding constant appears to be modulated by the pH of the solution. Spectroscopic measurements do not reveal any significant energy transfer between the Trp residues of β-lg and PPIX, however, fluorescence anisotropy decay measurements confirm the binding and the modulation introduced by the pH of the solution. Since β-lg has been shown to be stable within the range of pH adopted in our experiments (5.0–9.0), the results suggest that PPIX binds a site affected by the pH of the solution. Because of the crystallographic evidence an obvious site is near the aperture of the interior β-barrel however an alternative (or concurrent) binding site may still be present.

Keywords: Abbreviations; PPIX; Protoporphyrin IX; β-lg; β-lactoglobulin; GI; gastrointestinal tract; DMSO; Dimethylsulfoxide; KI; potassium iodide; S-V; Stern–Volmer; Trp; Tryptophan, TPPS; ₄; , meso-tetra(sulphonatophenyl)-porphine; ANS; 1-anilinonaphtalene-8-sulfonate; FRET; Fluorescence Resonance Energy Transfer; CD; Circular DichroismLactoglobulin; Porphyrin; Fluorescence spectroscopy; Binding

Conformation and stability of elastase from Atlantic cod, Gadus morhua by G. Jayaraman; S. Srimathi; J.B. Bjarnason (pp. 47-54).

Metal binding and conformational stability characteristics of psychrophilic elastase (ACE) from Atlantic cod ( Gadus morhua) has been investigated. Chelation to Ca²⁺ was found to be important for maintaining the biologically active conformation and for the thermal stability of the enzyme. However, presence of metal ions such as Zn²⁺, Fe³⁺ and Cu²⁺ was found to inhibit its hydrolytic activity and so did the chelating agent EDTA. Both pH and guanidinium chloride induced denaturation of the enzyme was followed by monitoring the changes in the tryptophan fluorescence. ACE exhibited a simple two-state unfolding pattern in both acidic and basic conditions with the midpoint of transition at pH values 4.08 and 10.29, respectively. Guanidinium chloride and heat induced denaturation of the enzyme was investigated at two pH values, 5.50 and 8.00, wherein the enzyme possesses similar tertiary structure but differ in its hydrolytic activity. Guanidinium chloride induced denaturation indicated that the enzyme unfolds with a C_m of 1.53 M at pH 8.0 and a Δ G_H2O of 6.91 kJ mol⁻¹ (28.65 J mol⁻¹ residue⁻¹) which is the lowest reported for psychrophilic enzymes investigated till-date. However, at pH 5.50, Δ G_H2O value is slightly lowered by 0.65 kJ mol⁻¹ consistent with the observed increase in the apparent quenching constant obtained with acrylamide. On the other hand, increase in T_m by 38.45 °C was observed for the enzyme at acid pH (5.50) in comparison to the heat induced unfolding at pH 8.0. The increase in the apparent T_m has been attributed to the possible weak intermolecular association of the enzyme molecules at moderately high temperatures that is favoured by the increase in the accessible surface area / dynamics under acidic conditions. The stability characteristics of ACE have been compared with the available data for mesophilic porcine pancreatic elastase and possible mechanism for the low temperature adaptation of ACE has been proposed.

Keywords: Elastase; Gadus morhua; Psychrophile; Denaturation; Stability

Why are HIV-1 fusion inhibitors not effective against SARS-CoV? Biophysical evaluation of molecular interactions by Salom� Veiga; Yunyun Yuan; Xuqin Li; Nuno C. Santos; Gang Liu; Miguel A.R.B. Castanho (pp. 55-61).

The envelope spike (S) glycoprotein of the severe acute respiratory syndrome associated coronavirus (SARS-CoV) mediates the entry of the virus into target cells. Recent studies point out to a cell entry mechanism of this virus similar to other enveloped viruses, such as HIV-1. As it happens with other viruses peptidic fusion inhibitors, SARS-CoV S protein HR2-derived peptides are potential therapeutic drugs against the virus. It is believed that HR2 peptides block the six-helix bundle formation, a key structure in the viral fusion, by interacting with the HR1 region. It is a matter of discussion if the HIV-1 gp41 HR2-derived peptide T20 (enfuvirtide) could be a possible SARS-CoV inhibitor given the similarities between the two viruses. We tested the possibility of interaction between both T20 (HIV-1 gp41 HR2-derived peptide) and T-1249 with S protein HR1- and HR2-derived peptides. Our biophysical data show a significant interaction between a SARS-CoV HR1-derived peptide and T20. However, the interaction is only moderate ( K_B=(1.1±0.3)×10⁵ M⁻¹). This finding shows that the reasoning behind the hypothesis that T20, already approved for clinical application in AIDS treatment, could inhibit the fusion of SARS-CoV with target cells is correct but the effect may not be strong enough for application.

Keywords: T20; Enfuvirtide; T-1249; Fusion inhibitor; HIV-1; SARS-CoV

Identification of BCOX1, a novel gene overexpressed in breast cancer by Jin Song; Wanjun Yang; Ie-Ming Shih; Zhen Zhang; Jining Bai (pp. 62-69).

The identification of tumor-associated antigens, which are specifically expressed in cancer tissues, is of utmost important for immunotherapy of breast cancer. We have combined in silico screening and experimental expression analysis to identify genes that are differentially expressed in breast carcinomas compared with their corresponding normal tissues. Using these approaches, we identified a novel gene, BCOX1, with overexpression in breast carcinoma. BCOX1 was highly homologous to KIAA0100, a hypothetical gene located on chromosome 17q11.2. RNA in situ hybridization shows that BCOX1 mRNA signal is mainly located in the cytoplasm of breast carcinoma epithelial cells, but not in those of normal epithelial cells, stroma cells and lymphocytes. Furthermore, mRNA expression of BCOX1 was moderately elevated in ductal in situ carcinoma (DCIS), peaked in invasive breast carcinoma (IBC) and metastatic breast carcinoma cells (MET) whereas absent in benign ductal epithelial cells. The predicted BCOX1 open reading frame of 666 bp encodes a putative protein of 222 amino acid residues with a calculated molecular weight of 24920 Da and a PI of 5.86. Computational analyses predict that the putative BCOX1 protein is a cytoplasmic protein. The functional relevance of this novel gene is yet to be determined. This study warrants further investigations to explore the molecular functions of BCOX1, and to determine its potential diagnostic and therapeutic applications for breast cancer.

Keywords: Abbreviations; BCOX1; breast cancer overexpressed gene 1; SAGE; serial analysis of gene expression; EST; expressed sequence tag; RT-PCR; reverse transcription-polymerase chain reactionSAGE; EST database; Gene characterization; Cancer

Inhibition of lipid peroxidation and protein oxidation in rat liver mitochondria by curcumin and its analogues by Qing-Yi Wei; Wei-Feng Chen; Bo Zhou; Li Yang; Zhong-Li Liu (pp. 70-77).

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione,1) is a yellow ingredient isolated from turmeric ( curcumin longa). It has been shown to exhibit a variety of biological activities including antioxidative activity. In order to find more active antioxidants with1 as the lead compound we synthesized curcumin analogues, i.e., 1-(3,4-dihydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (2), 1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-1,6-heptadiene-3,5-dione (3), 1,7-bis-(4-hydroxyphenyl)-1,6-heptadiene-3,5-dione (4), 1-(3,4-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (5), 1,7-bis(3,4-dimethoxyphenyl)-1,6-heptadiene-3,5-dione (6), and 1,7-diphenyl-1,6-heptadiene-3,5-dione (7), and evaluated their antioxidative activity. The in vitro oxidative damage to both lipids and proteins in rat liver mitochondria was used as a model to study the free radical-induced oxidative damage of biological lipids as well as proteins and the protective effects of these curcumin analogues. It was found that these compounds, except6 and7, could effectively inhibit the free radical induced lipid peroxidation and protein oxidative damage of rat liver mitochondria by H-atom abstraction from the phenolic groups. Compound 2 which bear ortho-diphenoxyl functionality exhibited remarkably higher antioxidative activity for lipids and proteins than curcumin and other analogues, and the 4-hydroxy-3-methoxyphenyl group also play an important role in the antioxidative activity.

Keywords: Abbreviations; Curcumin; 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione; AAPH; 2,2′-azobis(2-amidinopropane hydrochloride); TBA; Thiobarbituric acid; TBARS; thiobarbituric acid reactive substance; DPPH; 2,2-diphenyl-1-picrylhydrazyl; BHT; butylated hydroxytoluene; EDTA; sodium ethylenediaminetetraacetate; PBS; phosphate-buffered saline; TCA; trichloroacetic acid; DNPH; 2, 4-dinitrophenylhydrazine; SDS; Sodium dodecyl sulfateCurcumin; Phenolic antioxidant; Rat liver mitochondria; Antioxidation; Lipid peroxidation; Protein carbonyl; Structure/activity relationship

Intrinsic enoyl-CoA isomerase activity of rat acyl-CoA oxidase I by Jia Zeng; Guisheng Deng; Ding Li (pp. 78-85).

Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the β-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported. It was found that rat acyl-CoA oxidase I has intrinsic enoyl-CoA isomerase activity, which was confirmed using incubation followed with HPLC analysis in this study. Various 3-enoyl-CoA substrates with cis or trans configuration were synthesized and used in the study of enzyme substrate specificity. The isomerase activity of the enzyme was characterized through studies of kinetics, pH dependence, and enzyme inhibition. Most k_cat/ K_M values of rat peroxisomal acyl-CoA oxidase I for isomerization reaction are comparable with those of authentic rat liver peroxisomal Δ³-Δ²-enoyl-CoA isomerase and rat liver peroxisomal multifunctional enzyme 1 when hexenoyl-CoA and octenoyl-CoA with cis- or trans-configuration were used as substrate. Glu421 was found to be the catalytic residue for both oxidase and isomerase activities of the enzyme. The isomerase activity of rat peroxisomal acyl-CoA oxidase I is probably due to a spontaneous process driven by thermodynamic equilibrium with formation of a conjugated structure after deprotonation of substrate α-proton. The energy level of transition state may be lowered by a stable dienolate intermediate, which gain further stabilization via charge transfer with electron-deficient FAD cofactor of the enzyme.

Keywords: Abbreviations; ACD; acyl-CoA dehydrogenase; ACO; acyl-CoA oxidase; FAD; flavin adenine dinucleotide; MCAD; medium-chain acyl-CoA dehydrogenase; PCR; polymerase chain reaction; UV/Vis; ultraviolet-visible spectroscopyAcyl-CoA oxidase; Isomerase; β-oxidation; Fatty acid; Peroxisome; FAD

Neutrophil activation induced by the lectin KM+ involves binding to CXCR2 by Gabriela Pereira-da-Silva; Andr�a N. Moreno; Fabiana Marques; Constance Oliver; Maria C�lia Jamur; Ademilson Panunto-Castelo; Maria Cristina Roque-Barreira (pp. 86-94).

The lectin KM+ from Artocarpus integrifolia, also known as artocarpin, induces neutrophil migration by haptotaxis. The interactions of KM+ with both neutrophils and the extracellular matrix depend on the lectin's ability to recognize mannose-containing glycans. In the present study, we characterized the binding of KM+ to human neutrophils and the responses stimulated by this binding. Exposure to KM+ results in cell polarization, formation of a lamellipodium, and induction of deep ruffles on the cell surface. By fluorescence microscopy, we observed that KM+ is distributed homogeneously over the cell surface. KM+/ligand complexes are rapidly internalized, reaching maximum intracellular concentrations at 120 min, and decreasing thereafter. Furthermore, KM+ binding to the surface of human neutrophils is inhibited by the specific sugars,d-mannose or mannotriose. KM+-induced neutrophil migration is inhibited by pertussis toxin as well as by inhibition of CXCR2 activity. These results suggest that the KM+ ligand on the neutrophil surface is a G protein-coupled receptor (GPCR). The results also suggest that neutrophil migration induced by KM+ involves binding to CXCR2.

Keywords: Abbreviations:; CRD; carbohydrate recognition domain; CXCR-1; CXC-chemokine receptor type 1; CXCR-2; CXC-chemokine receptor type 2; ECM; extracellular matrix; FACS; fluorescence activated cell sorter; FITC; fluorescein isothiocyanate; fMLP; formyl-methionyl-leucyl-phenylalanine; GCP-2; granulocyte chemotactic protein-2; GPCR; G protein-coupled receptor; IL-8; interleukin-8; MAPK; mitogen-activated protein kinase; MNCF; macrophage-derived neutrophil chemotactic factor; PAGE; polyacrylamide gel electrophoresis; PLC; phospholipase C; PTx; pertussis toxin; PBS; phosphate-buffered saline; S.E.M.; standard error of the mean; SDS; sodium dodecyl sulfate; WGA; wheat germ agglutininLectin; Artocarpus integrifolia; Neutrophil; Cell migration; CXCR2 receptor; Inflammation

Relationship between toxicity of selected insecticides and expression of stress proteins (HSP, GRP) in cultured human cells: Effects of commercial formulations versus pure active molecules by Dalila Skandrani; Yolande Gaubin; Christian Vincent; Bernadette Beau; Jean Claude Murat; Jean-Pierre Soleilhavoup; Fran�oise Croute (pp. 95-103).

Three carbamate (formetanate, methomyl, pyrimicarb) and one pyrethroid (bifenthrin) insecticides were investigated both as pure chemicals and as commercial formulations in order to unveil possible toxic effects of additives and solvents present in the commercial formulations and to evaluate the cellular stress response as a defense mechanism. Toxic effects were evaluated on A549 cells, derived from a human lung carcinoma, by measuring (1) threshold concentrations leading to a decrease of the growth rate (LOEC), (2) sublethal concentrations (SC) which arrested growth without killing the cells, and (3) expression levels of several stress proteins, i.e., HSP27, HSP72/73, HSP90, GRP78, and GRP94. As compared to the pure active molecule, LOEC appeared at lower concentrations when using the commercial formulations, i.e., Dicarzol (formetanate), Lannate20 (methomyl) and Talstar or Kiros EV (bifenthrin). Propylene glycol and propylene glycol monomethyl ether, respectively, present in Talstar and kiros, do not account for the high toxicity of these commercial formulations and do not potentiate the toxicity of bifenthrin. Additive but not synergistic adverse effects were observed when cells are exposed to a mixture of 4 different commercial formulations. Our results show that the concentrations of active molecules recommended in floricultural general use or for spray preparations are much higher than SC concentrations, as determined on A549 pulmonary cells. GRP78 was up-regulated by all the insecticides, commercial preparations being more efficient to trigger the stress reaction. This suggests that insecticides and additives present in commercial formulations disrupt ER functions. Conversely, HSP72/73 was found to be down-regulated by all the insecticides. This seems to be related with a decrease of protein synthesis in the cytosol, as a result of the ER unfolded protein response. Indeed, tunicamycin, known to inhibit N-linked glycosylation in the ER, was found to induce a similar inverse correlation between GRP78 overexpression and HSP72/73 underexpression. Expression of GRP94 was found to be increased and HSP27 lowered by the highest concentrations of bifenthrin commercial formulations. Methomyl and Lannate20 only induced an underexpression of HSP90.

Keywords: Formetanate; Methomyl; Pyrimicarb; Bifenthrin; GRP78; HSP70

Antimicrobial action of novel chitin derivative by Jae-Young Je; Se-Kwon Kim (pp. 104-109).

Aminoethyl-chitin (AEC) was synthesized in an attempt to both increase solubility of chitin in water and biological activity. AEC was obtained by grafting 2-chloroethylamino hydrochloride onto chitin at C-6 position. The structure of AEC was elucidated FT-IR and¹H NMR spectroscopy, and its antimicrobial activity was investigated using three Gram-positive and Gram-negative bacteria. The integrity of the cell membranes of the representatives E. coli and S. aureus was investigated by determining the release of intracellular components of cells. When treated with AEC, release of 260 nm absorbing materials quickly increased both E. coli and S. aureus, but absorbance value was different due to the difference in cell structures. For detailed study, outer membrane (OM) and inner membrane (IM) permeabilization assay were performed using the fluorescent probe 1- N-phenylnaphthylamine (NPN) and the release of cytoplasmic β-galactosidase activity. The results showed that AEC rapidly increased NPN uptake and the release of cytoplasmic β-galactosidase via increasing the permeability of OM and IM. In addition, cytotoxic effect of AEC was assessed using human lung fibroblast (MRC-5) cell line, and AEC showed less toxic against MRC-5.

Keywords: Water-soluble chitin; Antimicrobial activity; Membrane permeabilization; Cytotoxicity

Characterization of FBX25, encoding a novel brain-expressed F-box protein by Olivier Hagens; Eleonora Minina; Susann Schweiger; Hans-Hilger Ropers; Vera Kalscheuer (pp. 110-118).

F-box proteins (FBPs) confer substrate specificity to the SCF-type (Skp1/Cul1/FBP) of ubiquitin ligase complexes through their F-box. Multiple FBPs have been predicted, but experimental evidence is lagging. We report on the predicted human FBP hFBX25 which we found to be disrupted in a mentally retarded translocation carrier suffering from epileptic seizures. We investigated hFBX25′s genomic organization and established hFBX25 as an FBP by verifying its interaction with Skp1 and Cul1. In the process, we identified an atypical serine residue in the F-box which is crucial for the hFBX25-Skp1 binding. We determined hFBX25′s subcellular localization. We found strong transcription in human brain. In mouse embryonic sections, mFbx25 shows predominantly neuronal expression and in adult mouse brain, expression is confined to the hippocampus, the cerebral cortex and the Purkinje cell layer. Interestingly, aberrations in the ubiquitin pathway have been linked to neurological conditions.

Keywords: FBX25; F-box protein; SCF; Neuronal expression; Ubiquitin pathway

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