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BBA - General Subjects (v.1770, #7)

Editorial Board (pp. ii).

Age-dependent expression of VEGF isoforms and receptors in the rabbit anterior cruciate ligament by Jochen G. Hofstaetter; Fawzy A. Saad; Ilse-Gerlinde Sunk; Klaus Bobacz; Ingeborg Friehs; Melvin J. Glimcher (pp. 997-1002).

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF. Those isoforms differ in biochemical and biological properties, and it has been reported that their expression patterns are tissue and age specific as well. We investigated the expression levels of VEGF isoforms (VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₃, VEGF₁₈₉) and its receptors (VEGFR-1, flt-1 and VEGFR-2, flk-1/KDR) in the anterior cruciate ligament (ACL) of 2- to 3-week-, 2-month-, and 18-month-old New Zealand White rabbits using Sybr green Real-Time RT-PCR. VEGF isoforms and both receptors were expressed in the ACL at all investigated ages. VEGF₁₂₁ was found to be the most abundant isoform at the ages under investigation, followed by VEGF₁₆₅, VEGF₁₈₉ and VEGF₁₈₃. All isoforms showed decreased expression levels with age, however the larger membrane bound isoforms, VEGF₁₈₃ and VEGF₁₈₉, showed the most striking age-associated decrease in expression level. VEGFR-1 expression levels increased with age, while the expression level of VEGFR-2 expression was highest at 2–3 weeks and was significantly lower at 2 and 18 months of age. Distinct age-associated differences in the expression level of VEGF isoforms as well as their receptors suggest differential physiological functions during development, maturation and ageing of the ACL.

Keywords: VEGF; Isoforms; Expression; Age-dependent; Anterior cruciate ligament; ACL; Knee; Rabbit

Protective role of unconjugated bilirubin on complement-mediated hepatocytolysis by Cecilia L. Basiglio; Sandra M. Arriaga; Héctor F. Pelusa; Adriana M. Almará; Marcelo G. Roma; Aldo D. Mottino (pp. 1003-1010).

Hyperbilirubinemia and complement-mediated immune attack on hepatocyte membrane are common features of certain hepatic diseases. To assess whether unconjugated bilirubin (UB) counteracts complement-mediated hepatocytolysis, we first generated a rabbit polyclonal antibody (Ab) against rat hepatocyte plasma membrane (RHPM). An assay performed with isolated rat hepatocytes in the presence of the polyclonal Ab and rat serum as complement donor demonstrated that UB inhibits cell lysis, as lactate dehydrogenase release into the medium was inhibited by the pigment in a dose-dependent manner. Immunofluorescence microscopy studies showed that UB significantly attenuates the binding of C3 to the hepatocyte–Ab complex. Further enzyme immunoassay studies showed that UB interferes the binding of C1q to purified anti-RHPM IgG, also in a dose-dependent manner. A dot-blot assay showed that [¹⁴C]-UB binds to C1q and human serum albumin (HSA) to a similar extent. A differential spectrum analysis of UB in the presence of C1q further confirmed that the pigment interacts with this protein. In conclusion, we demonstrated an inhibitory action of UB on complement-mediated Ab-induced hepatocytolysis, this action being evidenced at pathophysiological pigment concentrations (171 μM and higher). A direct binding of the pigment to C1q is likely involved.

Keywords: Hyperbilirubinemia; Classical complement pathway; Hepatocytes; Antibody; C1 complement

Quantitative study of the drug efflux kinetics from sensitive and MDR human breast cancer cells by Chenguang Zhou; Peng Shen; Yiyu Cheng (pp. 1011-1020).

Cancer multidrug resistance (MDR) is a major impediment to effective chemotherapy in human cancer, in which P-glycoprotein and Multidrug Resistance-Associated protein figure prominently. Design and exploitation of novel clinical MDR inhibitors is greatly hindered by a lack of understanding of drug efflux dynamics in drug-sensitive and resistant cells. The aim of our study was to provide a microelectrode method for measuring the multidrug transporter mediated efflux of doxorubicin as well as a corresponding data analysis method for quantifying the efflux kinetic parameters. We performed experiments using carbon fiber microelectrode to detect doxorubicin efflux from a monolayer of human breast cancer MCF-7 cells and derived MDR cells (MCF-7/ADR), established a material transport model and proposed a novel inverse method to quantitatively characterize the diffusion dynamics. The kinetic parameters of doxorubicin efflux from MCF-7 and MCF-7/ADR cells in the presence or absence of MDR inhibitors were estimated. Our investigations showed the average initial doxorubicin efflux rate of MCF-7/ADR that was 5.2 times faster than of MCF-7. After treatment by tetramethylpyrazine or verapamil, the drug efflux rate of the MCF-7/ADR cells was reduced by about half that of those without inhibitors. The novel methodology presented suggests new and expanded applications for computer-aided reconstruction of the drug efflux process, microelectrode design, and high-throughput drug screening.

Keywords: Abbreviations; MDR; multidrug resistance; CF; carbon fiber; CFME; carbon fiber microelectrode; P-gp; P-glycoprotein; MRP; Multidrug Resistance-Associated protein; ABC; members of the ATP-binding cassette; Dox; doxorubicin (also called Adriamycin); TMP; tetramethylpyrazine; VRP; verapamil; HVC; hydrophobic vacuum-cleanerHuman breast cancer; Multidrug resistance; Kinetics parameter; Mathematical model; Electrochemical measurement

Suppression of 5-lipoxygenase gene is involved in triptolide-induced apoptosis in pancreatic tumor cell lines by G.X. Zhou; X.L. Ding; J.F. Huang; H. Zhang; S.B. Wu (pp. 1021-1027).

Pancreatic adenocarcinoma is characterized by a poor prognosis and lack of response to conventional therapy. The purpose of this study was to investigate the effects of triptolide (TL) on proliferation and apoptosis of pancreatic cancer cells in vitro. We found that TL induced prominent growth inhibition and apoptosis in human pancreatic cell lines. In addition, TL treatment significantly down-regulated 5-lipoxygenase (5-LOX) expression, as well as downstream leukotriene B4 (LTB4) production, in these cell lines. Furthermore, overexpression of 5-LOX in SW1990 cell lines or exogenous LTB4 made them more resistant to TL-induced apoptosis, which was correlated with increased Bcl-2 expression. Taken together, these findings suggest that inhibition of the 5-LOX pathway of arachidonic acid metabolism is associated with the anti-proliferation activity of TL. We also provide evidence that TL has clinical therapeutic value for patients with pancreatic cancer.

Keywords: Abbreviations; TL; triptolide; 5-LOX; 5-lipoxygenase; LTB4; leukotriene B4Triptolide; Pancreatic tumor; 5-lipoxygenase; Caspase-3; Bcl-2

Modulations of the calcineurin/NF-AT pathway in skeletal muscle atrophy by Paola Costelli; Vanessa Almendro; Maria Teresa Figueras; Patrizia Reffo; Fabio Penna; Manuela Aragno; Raffaella Mastrocola; Giuseppe Boccuzzi; Silvia Busquets; Gabriella Bonelli; Francisco J. Lopez Soriano; Josep M. Argilés; Francesco M. Baccino (pp. 1028-1036).

Calcineurin has been proposed to regulate skeletal muscle hypertrophy, while its relevance to the pathogenesis of muscle atrophy is unknown. The present study was aimed to investigate if perturbations of the calcineurin pathway may be involved in causing skeletal muscle atrophy in two different experimental conditions: cancer cachexia (rats bearing the AH-130 hepatoma), and hyperglycemia (rats treated with streptozotocin). Calcineurin expression in the gastrocnemius was comparable between tumor hosts and controls. By contrast, besides unchanged calcineurin mRNA levels, those of protein were lower in diabetic animals than in controls. The DNA-binding activity of the transcription factors NF-AT and MEF-2 was analysed as an indirect measure of calcineurin activity in vivo. The nuclear translocation of both factors was similar in tumor hosts and controls. Consistently with the reduced calcineurin protein levels, NF-AT DNA-binding activity significantly decreased in the gastrocnemius of diabetic rats compared to controls. Finally, muscle wasting correction afforded in the AH-130 hosts by pentoxifylline or interleukin-15 was not paralleled by changes of calcineurin mRNA levels, while treatment of diabetic animals with dehydroepiandrosterone partially prevented calcineurin down-regulation. These results suggest that modulations of calcineurin activity may be involved in the pathogenesis of muscle wasting in diabetes though not in cancer cachexia.

Keywords: Cancer cachexia; Calcineurin; Diabetes; Muscle wasting; NF-AT

A novel extracellular EF-hand protein involved in the shell formation of pearl oyster by Jing Huang; Cen Zhang; Zhuojun Ma; Liping Xie; Rongqing Zhang (pp. 1037-1044).

Mollusk shell formation is a complicated and highly controlled calcium metabolism process. Previous studies revealed that several EF-hand calcium-binding proteins actively participate in the regulation of shell mineralization. In this study, we cloned a full-length cDNA encoding a novel extracellular EF-hand calcium-binding protein (named EFCBP) from the pearl oyster, Pinctada fucata, according to the EF-hand motifs of calmodulin. Although it shares high similarity with the calmodulin family in its EF-hand signatures, EFCBP just has two EF-hand motifs and belongs to a new separate group from the other EF-hand proteins according to a phylogenetic analysis. EFCBP is specifically expressed in shell mineralization-related tissues, viz. the mantle, the gill, and the hemocytes. Moreover, its expression responds quickly only to the shell damage, but not to the damage of other tissues and the infection of the lipopolysaccharides from Escherichia coli. These results suggest that EFCBP might be an important regulator of shell formation. This finding may help better understand the functions of EF-hand proteins on the regulation of mollusk shell formation.

Keywords: Abbreviations; CaM; calmodulin; CaLP; calmodulin-like protein; PFMG1; Pinctada fucata; mantle gene 1; PFMG6; Pinctada fucata; mantle gene 6; RACE; rapid amplification of cDNA ends; GAPDH; Glyceraldehyde 3-phosphate dehydrogenase; DEPC; diethypyrocarbonate; LPS; lipopolysaccharides; BLAST; basic local alignment search toolEF-hand; Biomineralization; Calcium metabolism; Regulator; EFCBP; Pinctada fucata

Expression and characterization of cytochrome P450 2X1 in channel catfish ( Ictalurus punctatus) by Sasan Mosadeghi; Bjarte Furnes; Aline Y.O. Matsuo; Daniel Schlenk (pp. 1045-1052).

Previous studies in channel catfish identified a novel cDNA encoding the cytochrome P450 isoform, CYP2X1. To characterize the substrate specificity of CYP2X1, the 57 kDa protein was expressed in Sf9 cells. Microsomes from Sf9 cells transfected with CYP2X1 demonstrated a maximum carbon monoxide-reduced difference spectrum at 450 nm and catalyzed aminopyrine and benzphetamine demethylase activity with catalytic efficiency (Vmax/Km) values of 0.82 pmol/nmol P450/min and 4.39 pmol/nmol P450/min, respectively. However, enzymatic activity was not observed following incubation with p-nitrophenol, benzyloxyresorufin or pentoxyresorufin. Expression of CYP2X1 transcription was significantly elevated in the gills and liver relative to that detected in brain, kidney and heart. In the brain, liver and heart, intraperitoneal injections with clofibric acid, ethanol, pyridine and rifampin failed to alter expression of CYP2X1 mRNA. In kidney, pyridine significantly suppressed the expression of CYP2X1 transcription ( p≤0.05). These results indicate CYP2X1 displays minimal catalytic activities consistent with other piscine CYP2 isoforms, and unique tissue expression and regulation patterns in juvenile channel catfish.

Keywords: Cytochrome P450; Catfish; Benzphetamine; Pyridine

Selenocysteine β-lyase and methylselenol demethylase in the metabolism of Se-methylated selenocompounds into selenide by Kazuo T. Suzuki; Kazuki Kurasaki; Noriyuki Suzuki (pp. 1053-1061).

The lyase activity toward Se-methylated selenoamino acids and the demethylase activity toward methylselenol in the metabolism of selenium were characterized in vitro. The β- and γ-lyase activities toward selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys), respectively, were compared under exactly identical conditions by incubating⁷⁷Se-SeMet and⁷⁶Se-MeSeCys simultaneously in a liver supernatant, and then estimated by the decreases in the labeled starting selenoamino acids (MeSeCys and SeMet), and also by the increases in the labeled enzyme products (methylselenol and selenide) after oxidation to methylseleninic acid (MSA^IV) and selenite, respectively, by HPLC-inductively coupled plasma-mass spectrometry (ICP-MS). Only⁷⁶Se-MeSeCys was decreased and only⁷⁶Se-selenite was produced, suggesting that conversion of MeSeCys to methylselenol by β-lyase followed by that of methylselenol to selenide by demethylase actively occurred in the liver supernatant. The demethylase activity was characterized by incubating⁷⁷Se-methylselenol produced in situ from⁷⁷Se-MSA^IV and glutathione in a partially purified enzyme preparation. It was found that demethylation takes place directly through an attack by a hydroxide anion on the methyl group of methylselenol producing selenide and methanol, selenide being detected on HPLC-ICP-MS after oxidation to selenite, and methanol on GC-MS. It was concluded that β- but not γ-lyase activity could be detected in a liver supernatant, and that the resulting methylselenol product is demethylated through hydrolysis, with methanol and selenide being produced (MeSeCys→CH₃SeH→HSeH+CH₃OH).

Keywords: Abbreviations; DMSe; dimethylselenide; DTT; dithiothreitol; GSH; glutathione; ICP-MS; inductively coupled plasma-mass spectrometry; MSA; ^IV; methylseleninic acid; MeSeCys; Se-methylselenocysteine; SeMet; selenomethionine; TMSe; trimethylselenoniumSelenium; Methylselenol; Demethylase; β-lyase; Selenide; Methylselenocysteine; Methylseleninic acid; Selenomethionine; Speciation

Linoleoyl lysophosphatidic acid and linoleoyl lysophosphatidylcholine are efficient substrates for mammalian lipoxygenases by Long Shuang Huang; Mee Ree Kim; Tae-Sook Jeong; Dai-Eun Sok (pp. 1062-1070).

Oxygenation of two lysophospholipids, 1-linoleoyl lysophosphatidylcholine (linoleoyl-lysoPC) and 1-linoleoyl lysophosphatidic acid (linoleoyl-lysoPA), by reticulocyte lipoxygenase (LOX) or porcine leukocyte LOX was measured by monitoring the formation of conjugated dienes. Consistent with the above, the formation of linoleoyl-lysophospholipid hydroperoxide as oxygenation product was confirmed by LC/MS analyses. In further study, the oxygenation products of linoleoyl-lysoPC or linoleoyl-lysoPA were found to contain hydroperoxide group predominantly at C-13 with the S enantiomer as a major one, in a good agreement with the positional-specificity and stereo-selectivity of reticulocyte LOX or leukocyte LOX in oxygenation of linoleic acid. The kinetic study indicates that linoleoyl-lysoPA and linoleoyl-lysoPC are no less efficient than linoleic acid as substrates of reticulocyte LOX as well as leukocyte LOX. In contrast, these lysophospholipids were not oxygenated efficiently by potato LOX. Thus, linoleoyl-lysophospholipids such as linoleoyl-lysoPA or linoleoyl-lysoPC could be utilized as efficient substrates for some mammalian lipoxygenases.

Keywords: Abbreviations; linoleoyl-lysoPC; 1-linoleoyl lysophosphatidylcholine; linoleoyl-lysoPA; 1-linoleoyl lysophosphatidic acid; LOX; lipoxygenase; HODE; hydroxyoctadecadienoic acid; DLPC; dilinoleoyl phosphatidylcholine; SP; straight-phase; RP; reverse phaseReticulocyte; Leukocyte; Lipoxygenase; Linoleoyl-lysophosphatidylcholine; Linoleoyl-lysophosphatidic acid

Molecular recognition of DNA by small molecules: AT base pair specific intercalative binding of cytotoxic plant alkaloid palmatine by Kakali Bhadra; Motilal Maiti; Gopinatha Suresh Kumar (pp. 1071-1080).

The base dependent binding of the cytotoxic alkaloid palmatine to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA–dT).poly(dA–dT), poly(dG).poly(dC) and poly(dG–dC).poly(dG–dC) was examined by competition dialysis, spectrophotometric, spectrofluorimetric, thermal melting, circular dichroic, viscometric and isothermal titration calorimetric (ITC) studies. Binding of the alkaloid to various polynucleotides was dependent upon sequences of base pairs. Binding data obtained from absorbance measurements according to neighbour exclusion model indicated that the intrinsic binding constants decreased in the order poly(dA).poly(dT)>poly(dA–dT).poly(dA–dT)>poly(dG–dC).poly(dG–dC)>poly(dG).poly(dC). This affinity was also revealed by the competition dialysis, increase of steady state fluorescence intensity, increase in fluorescence quantum yield, stabilization against thermal denaturation and perturbations in circular dichroic spectrum. Among the polynucleotides, poly(dA).poly(dT) showed positive cooperativity at binding values lower than r=0.05. Viscosity studies revealed that in the strong binding region, the increase of contour length of DNA depended strongly on the sequence of base pairs being higher for AT polymers and induction of unwinding–rewinding process of covalently closed superhelical DNA. Isothermal titration calorimetric data showed a single entropy driven binding event in the AT homo polymer while that with the hetero polymer involved two binding modes, an entropy driven strong binding followed by an enthalpy driven weak binding. These results unequivocally established that the alkaloid palmatine binds strongly to AT homo and hetero polymers by mechanism of intercalation.

Keywords: Abbreviations; DNA; deoxyribonucleic acid; CD; circular dichroism spectroscopy; CCS Col E; ₁; DNA; covalently closed superhelical DNA; ITC; isothermal titration calorimetry; Tm; thermal melting temperature; D/P; alkaloid/ DNA nucleotide phosphate; P/D; DNA nucleotide phosphate/alkaloid molar ratio; ESI-MS; electrospray ionization mass spectroscopy; poly(A); polyadenylic acid; AT; adenine-thymine; GC; guanine-cytosinePalmatine; Polynucleotides; Spectroscopy; Viscosity; Calorimetry; AT specific binding; Intercalation

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BBA - General Subjects (v.1770, #7)