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BBA - General Subjects (v.1760, #10)

Editorial Board (pp. ii).

Strain dependence of the cell wall-damage induced stimulon in Staphylococcus aureus by N. McCallum; G. Spehar; M. Bischoff; B. Berger-Bächi (pp. 1475-1481).

The vancomycin stress induced transcriptome of the methicillin susceptible Staphylococcus aureus (MSSA) strain Newman was determined by microarray analysis. Subsets of the induced ORFs corresponded to those previously reported to be induced by vancomycin in the methicillin resistant S. aureus (MRSA) strains N315 and JH1, and/or by other cell wall active antibiotics in RN450; while other ORFs appeared to be induced strain specifically in Newman. Northern analyses showed that the induction pathway for several of the ORFs appeared to be altered in a number of clinical NARSA isolates. Induction was found to be dependent on inhibitory concentrations of antibiotics.

Keywords: S. aureus; Glycopeptide intermediate resistance; Cell wall-stress stimulon

Vitamin K deficiency reduces testosterone production in the testis through down-regulation of the Cyp11a a cholesterol side chain cleavage enzyme in rats by Hitoshi Shirakawa; Yusuke Ohsaki; Yoshihiko Minegishi; Naofumi Takumi; Kousaku Ohinata; Yuji Furukawa; Takeo Mizutani; Michio Komai (pp. 1482-1488).

Vitamin K (K) is an essential factor for the posttranslational modification of blood coagulation factors as well as proteins in the bone matrix (Gla proteins). It is known that K is not only distributed in the liver and bones but also abundantly distributed in the brain, kidney, and gonadal tissues. However, the role of K in these tissues is not well clarified. In this study, we used DNA microarray and identified the genes whose expression was affected in the testis under the K-deficient (K-def) state. The expression of genes involved in the biosynthesis of cholesterol and steroid hormones was decreased in the K-def group. The mRNA levels of Cyp11a – a rate-limiting enzyme in testosterone synthesis – positively correlated with the menaquinone-4 (MK-4) concentration in the testis. Moreover, as compared to the control (Cont) and K-supplemented (K-sup) groups, the K-def group had decreased testosterone concentrations in the plasma and testis. These results suggested that K is involved in steroid production in the testis through the regulation of Cyp11a.

Keywords: Vitamin K; Testis; Testosterone; Steroidogenesis; Cyp11a

Use of novel cationic bile salts in cholesterol crystallization and solubilization in vitro by Shreedhar Bhat; Diana Leikin-Gobbi; Fred M. Konikoff; Uday Maitra (pp. 1489-1496).

Unnatural bile salts have been synthesized with a cationic group at the side chain of natural bile acids. These cationic bile salts aggregate in water and aqueous salt solutions in a manner similar to their natural counterparts. The critical micellar concentrations of the cationic bile salts were measured using a fluorescence method. Cationic bile salts aggregated at a concentration lower than natural deoxycholic acid. Since dihydroxy bile salt micelles are well known for cholesterol dissolution/removal, the dissolution in the cationic micelles has been evaluated. The cationic analogs dissolve approximately 70 mg/dL of cholesterol, which is comparable to taurochenodeoxycholate micelle under identical bile salt concentrations. Cholesterol dissolution in cationic bile salt micelle enhanced upon adding various amounts of PC. Cholesterol crystallization was studied in model bile at various cationic bile salt concentrations. The addition of 5, 15 and 30 mM of the cationic bile salts attenuated the crystallization process, without influencing the crystal observation time or decreasing the final amount of crystals formed. All these effects were comparable to those observed with cholic acid. These findings suggest that cationic bile salts have physico-chemical properties analogous to those of natural anionic bile salts, and thus may have therapeutic potential.

Keywords: Cationic bile salt; Cationic micelle; log; P; value; Pyrene probe; Cholesterol dissolution; Cholesterol crystallization

Human pancreatic lipase-related protein 2: Tissular localization along the digestive tract and quantification in pancreatic juice using a specific ELISA by Cécilia Eydoux; Ahmed Aloulou; Josiane De Caro; Philippe Grandval; René Laugier; Frédéric Carrière; Alain De Caro (pp. 1497-1504).

Human pancreatic lipase-related protein 2 (HPLRP2) was previously found to be secreted by the exocrine pancreas. HPLRP2 shows a high level of activity on galactolipids, and might be involved in the digestion of these common vegetable lipids. Specific antibodies were raised in rabbits using a synthetic HPLRP2 peptide selected for its weak amino acid homology with the corresponding peptides of classical human pancreatic lipase (HPL) and human pancreatic lipase-related protein 1 (HPLRP1). ELISA and Western blotting data showed that these antibodies did not react with HPL or HPLRP1. Various tissues from the digestive tract were subjected to Western blotting analysis with the specific anti-peptide HPLRP2 antibody and the expression of HPLRP2 was detected in the pancreas and colon. An ELISA was developed for specifically measuring the HPLRP2 levels in pure pancreatic juice. This procedure was performed using the anti-peptide HPLRP2 antibody as the captor antibody and a biotinylated anti-HPLRP2 polyclonal antibody as the detector antibody. The lowest HPLRP2 quantification limit was found to be 50 μg/L and the reference range for the present assay was 50 μg–500 μg/L. HPL and HPLRP2 levels were measured using specific ELISAs in pancreatic juice from patients with and without pancreatic disorders. Patients with chronic calcifying pancreatitis (CCP) had significantly lower levels of both HPL and HPLRP2 than the controls subjects. The mean HPLRP2 to HPL ratio was estimated to be 28.30% (w/w) and 23.96% (w/w) in controls subjects and CCP patients, respectively, and the difference was not significant. The levels of HPL and HPLRP2 are therefore similarly reduced in both healthy patients and CCP patients.

Keywords: Classical pancreatic lipase; Pancreatic lipase-related protein 2; Pancreatic juice; Polyclonal antibody; Anti-peptide antibody; ELISA

Ursolic acid and its derivative inhibit protein tyrosine phosphatase 1B, enhancing insulin receptor phosphorylation and stimulating glucose uptake by Wei Zhang; Di Hong; Yueyang Zhou; Yinan Zhang; Qiang Shen; Jing-ya Li; Li-hong Hu; Jia Li (pp. 1505-1512).

Protein tyrosine phosphatase 1B (PTP1B) is a key element in the negative regulation of the insulin signaling pathway and may play an important role in diabetes and obesity. We identified ursolic acid, a natural pentacyclic triterpenoid that occurs widely in traditional Chinese medicinal herbs, as an inhibitor of PTP1B by screening an extract library of the traditional Chinese medicinal herbs used a diabetes clinic. By modifying urosolic acid, we designed and synthesized a derivative with a K_i of 283 nM. As competitive inhibitors of PTP1B, ursolic acid and its derivative also inhibit T-cell protein tyrosine phosphatase and src homology phosphatase-2 but not leucocyte antigen-related phosphatase or protein tyrosine phosphatase α and ε, which are all possibly involved in the insulin pathway. The ursolic acid derivative enhanced insulin receptor phosphorylation in CHO/ hIR cells and stimulate glucose uptake in L6 myotubes.

Keywords: PTP1B; Inhibitor; Ursolic acid; Traditional Chinese medicinal herb; Derivative; Competitive; Insulin receptor; Glucose uptake

Transport of liposomal and albumin loaded curcumin to living cells: An absorption and fluorescence spectroscopic study by Amit Kunwar; Atanu Barik; Ruchi Pandey; K. Indira Priyadarsini (pp. 1513-1520).

Curcumin, a lipid soluble antioxidant, exhibits solvent and medium sensitive absorption and fluorescence properties. Using such changes, the average binding constants of curcumin to phosphatidylcholine (PC) liposomes and human serum albumin (HSA) were estimated to be 2.5×10⁴ M−¹ and 6.1×10⁴ M−¹ respectively. From the studies on temperature dependent fluorescence anisotropy of liposomal curcumin and its fluorescence quenching by acrylamide and iodide, it was concluded that curcumin is located in the gel phase of the liposomes. Similarly from the studies on quenching of tryptophan fluorescence in HSA by curcumin, it was found to be in the same domain as that of tryptophan. Both liposomal and HSA vehicles were examined for the transfer of curcumin to spleen lymphocyte cells, EL4 lymphoma cell line and compared with aqueous DMSO vehicles. From these studies it was found that liposomal vehicle is capable of loading more curcumin in to cells than HSA or aqueous-DMSO, and lymphoma cells show preferential uptake of curcumin to lymphocytes. The fluorescence of curcumin in EL4 lymphoma cells was found to be significantly higher as compared to the lymphocytes. The present study demonstrates a simple and quantitative method of estimation of curcumin delivered to cells by different vehicles using absorption and fluorescence spectroscopy.

Keywords: Curcumin; Liposomes; Human serum albumin; Absorption spectra; Fluorescence; Lymphocytes; EL4 lymphoma cells; Uptake

Temporomandibular joint synovial fibroblasts mediate serine proteinase dependent Type I collagen degradation by F. Song; A.S. Bergdoll; L.J. Windsor (pp. 1521-1528).

Degenerative temporomandibular joint (TMJ) disorders are characterized by the excessive turnover of collagen. In addition to the matrix metalloproteinase (MMP) and cathepsin mediated collagen degradation pathways, a serine proteinase dependent pathway has recently been identified in TMJ fibroblasts. This study focused on further characterizing this serine proteinase pathway utilizing a media-mediated collagen degradation assay and zymography. The conditioned media from cell-mediated collagen degradation assays were incubated with Type I collagen at pH 7.5 with or without a MMP inhibitor (1,10-phenanthroline), serine proteinase inhibitors (α1-antitrypsin and soybean trypsin inhibitor, STI), or cysteine proteinase inhibitors. The data showed that 1,10-phenanthroline and STI reduced the collagen cleavage by 12.33% and 47.78%, respectively. The cysteine proteinase inhibitors had no effect. The combination of α1-antitrypsin and 1,10-phenanthroline inhibited the cleavage by 79.22%, while STI and 1,10-phenanthroline together blocked the cleavage by 85.44%. Zymography identified a proteinase at approximate 22.5 kDa, which was more effectively blocked by serine proteinase inhibitors than by MMP or cysteine proteinase inhibitors. Reverse transcript-PCR and real-time PCR results demonstrated that TMJ cells did not express trypsinogen-2 mRNA, a collagen cleaving serine proteinase. This study demonstrated that TMJ fibroblasts can predominantly utilize a serine proteinase to mediate collagen degradation, which is not trypsinogen-2.

Keywords: Serine Proteinase; Synovial Fibroblast; Collagen Degradation

Ca²⁺-transport in sea urchin unfertilized eggs: Regulation by endogenous sulfated polysaccharides and K⁺ by Ana M. Landeira-Fernandez; Rafael S. Aquino; Paulo A.S. Mourão; Leopoldo de Meis (pp. 1529-1535).

Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca²⁺ by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K⁺ leading to an enhancement of the Ca²⁺ transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca²⁺ at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K⁺ but not by Li⁺ and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K⁺. These data suggest that the regulation of the SERCA pump by K⁺ and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.

Keywords: Ca; ²⁺; -transport; Endoplasmic reticulum; K; ⁺; activation; SERCA; Sea urchin egg; Sulfated polysaccharide

Antiproliferative and apoptosis inducing effect of lactoferrin and black tea polyphenol combination on hamster buccal pouch carcinogenesis by Kurapathy Venkata Poorna Chandra Mohan; Halagowder Devaraj; Duvuru Prathiba; Yukihiko Hara; Siddavaram Nagini (pp. 1536-1544).

Combination chemoprevention using tea polyphenols as one of the components has received growing consideration in recent years. The present study was designed to evaluate the antiproliferative and apoptosis inducing effects of bovine lactoferrin (bLF) and black tea polyphenol (Polyphenon-B: P-B) combination on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Topical application of DMBA for 14 weeks induced buccal pouch tumours that showed aberrant expression of cytokeratins, a marker for epithelial carcinomas. This was associated with increased cell proliferation and evasion of apoptosis as revealed by upregulation of proliferating cell nuclear antigen, NF-κB, mutant p53, Bcl-2 and downregulation of Bax, Fas and caspase 3 protein expression. Although dietary administration of bLF and Polyphenon-B alone significantly reduced tumour incidence, combined administration of bLF and Polyphenon-B was more effective in inhibiting HBP carcinogenesis by restoring normal cytokeratin expression, inhibiting cell proliferation and inducing apoptosis. These findings suggest that a “designer item�? approach will be useful for human oral cancer prevention strategies.

Keywords: Abbreviations; bLF; bovine lactoferrin; CKs; cytokeratins; DMBA; 7,12-dimethyl-benz[a]anthracene; P-B; Polyphenon-B; PCNA; proliferating cell nuclear antigenApoptosis; Black tea polyphenols; Combination chemoprevention; Cytokeratins; Hamster buccal pouch carcinogenesis; Cell proliferation

The specific, submicromolar- K_m ADP-ribose pyrophosphatase purified from human placenta is enzymically indistinguishable from recombinant NUDT9 protein, including a selectivity for Mn²⁺ as activating cation and increase in K_m for ADP-ribose, both elicited by H₂O₂ by António Carloto; María Jesús Costas; José Carlos Cameselle; Alexander G. McLennan; João Meireles Ribeiro (pp. 1545-1551).

Free ADP-ribose is a putative second messenger and also a potentially toxic compound due to its non-enzymic reactivity towards protein side chains. ADP-ribose hydrolysis is catalysed by NDP-sugar/alcohol pyrophosphatases of differing specificity, including a highly specific, low- K_m ADP-ribose pyrophosphatase. In humans, a submicromolar- K_m ADP-ribose pyrophosphatase has been purified from placenta, while recombinant NUDT9 has been described as a similarly specific enzyme with a nudix motif, but with a 10²–10³ higher K_m. Here, a comparative study of both proteins is presented showing that they are in fact enzymically indistinguishable; crucially, they both have submicromolar K_m for ADP-ribose. This study firmly supports the view that the ADP-ribose pyrophosphatase present in human tissues is a product of the NUDT9 gene. In addition, this study reveals previously unknown properties of both enzyme forms. They display the same, differential properties in the presence of Mg²⁺ or Mn²⁺ as activating cations with respect to substrate specificity, ADP-ribose saturation kinetics, and inhibition by fluoride. Treatment with H₂O₂ alters the Mg²⁺/Mn²⁺ responses and increases the K_m values for ADP-ribose, changes that are reversed by DTT. The results are discussed in relation to the proposed roles for ADP-ribose in oxidative/nitrosative stress and for ADP-ribose pyrophosphatase as a protective enzyme whose function is to limit the intracellular accumulation of ADP-ribose.

Keywords: Abbreviations; DTT; dl; -dithiothreitol; FPC; full progress curve (assay); GSSG; oxidized glutathione; IRD; initial rate discontinuous (assay); NAPQI; N; -acetyl-; p; -benzoquinoneimine; NDP; nucleoside 5′-diphosphate; TRPM; transient receptor potential melastatinAdenosine diphosphate ribose; Adenosine diphosphate ribose pyrophosphatase; Hydrogen peroxide; Nudix hydrolase; Oxidative stress

Protein kinase Cβ isoform down-regulates the expression of MDR3 P-glycoprotein in human Chang liver cells by Satoshi Suzuki; Hisao Hayashi; Kenji Takagi; Takaaki Kondo; Kenzo Takagi; Jun Ueyama; Shinya Wakusawa (pp. 1552-1557).

The MDR3 protein is a transporter of phosphatidylcholine on the canalicular membrane of human hepatocytes. Previously we showed that the expression of MDR3 mRNA was down-regulated by phorbol 12-myristate 13-acetate (PMA) in human Chang liver cells. In the present study, to elucidate the isoform of protein kinase C (PKC), which influences the level of MDR3 protein, we investigated the effects of PKC-specific inhibitors and antisense oligonucleotides. The level of protein decreased around 50% after treatment for 3–5 days using the dosage of PMA effective against the mRNA expression. The half-life of the MDR3 protein was estimated to be about 5 days. This decrease was antagonized by GF109203X, a non-selective inhibitor of PKCs, and Gö6976, a selective inhibitor for PKCα/β. These inhibitors also suppressed the reduction in MDR3 protein. To specify the isoform of PKC, the cells were treated with antisense oligonucleotide of PKCα or PKCβ. The suppressive effects on MDR3 mRNA of PMA were attenuated in antisense PKCβ-treated cells, but those in antisense PKCα-treated cells were not attenuated. These suggested that PKCβ plays a regulatory role in the expression of MDR3.

Keywords: Abbreviations; MDR; multidrug resistance; P-gp; P-glycoprotein; PCR; polymerase chain reaction; PMA; phorbol 12-myristate 13-acetate; DMSO; dimethylsulfoxideMDR3; PMA; PKCβ; GF109203X; Gö6976; Chang liver cells

Evaluation of the dietary effects of coenzyme Q in vivo by the oxidative stress marker, hydroxyoctadecadienoic acid and its stereoisomer ratio by Yasukazu Yoshida; Mieko Hayakawa; Yoko Habuchi; Etsuo Niki (pp. 1558-1568).

Coenzyme Q (CoQ) is an endogenous enzyme cofactor that may provide protective benefits as an antioxidant. In this study, in order to determine whether the concentrations of CoQ₉ are associated with the oxidative status in vivo, the effects of dietary supplements of CoQ₉ on mice were evaluated by using a new biomarker, total hydroxyoctadecadienoic acid (tHODE). Biological samples were first reduced and then saponified to convert the various oxidation products of linoleates to tHODE. Subsequently, by using GC-MS analyses, we simultaneously determined the absolute concentration of tHODE; its stereoisomer ratio, 9- and 13- (Z,E)-HODE/9- and 13- (E,E)-HODE, which is a measure of the hydrogen donor capacity of antioxidants; and the concentration of 8-iso-prostaglandin F_2α (8-iso-PGF_2α). Remarkable decreases in tHODE and 8-iso-PGF_2α levels were observed in the plasma, erythrocytes, liver, and brain of mice that were maintained for 1 month on an α-tocopherol (αT)-free (E-free) diet supplemented with ubiquinone-9 (Q₉; 0.04 wt.%) as compared to those of mice that were fed an E-free diet. The ( Z,E/E,E) HODE ratio was increased in the plasma and erythrocytes of mice that were fed a Q₉-fortified diet as compared to those that were fed an E-free diet. In particular, the ( Z,E/E,E) HODE ratios in the plasma and brain were significantly correlated with the concentrations of ubiquinol-9 (Q₉H₂). Further, the liver and brain levels of tHODE and 8-iso-PGF_2α were significantly correlated with the plasma and erythrocyte levels of tHODE and 8-iso-PGF_2α, respectively, and in some cases, also exhibited significant correlations with antioxidants. These results indicate that the plasma and erythrocyte levels of tHODE and its stereoisomeric ratio can be prominent biomarkers for the evaluation of the oxidative status and antioxidant capacity in vivo, including in the liver and brain, and that CoQ plays a major role in the in vivo antioxidant network.

Keywords: Abbreviations; BSTFA; N,O-bis(trimethylsilyl)trifluoroacetamide; CoQ; coenzyme Q; GPT; glutamic pyruvic transaminase; tHODE; total hydroxyoctadecadienoic acid; HPODE; hydroperoxyoctadecadienoic acid; 8-iso-PGF; _2α; 8-iso-prostaglandin F; _2α; PBS; phosphate-buffered saline; αT; α-tocopherol; TBARS; thiobarbituric acid reactive substancesCoenzyme Q; Total hydroxyoctadecadienoic acid; Antioxidant capacity; Stereo-isomer ratio; Isoprostane

N-linked glycosylation sites affect secretion of cryptococcal phospholipase B1, irrespective of glycosylphosphatidylinositol anchoring by Kylie M. Turner; Lesley C. Wright; Tania C. Sorrell; Julianne T. Djordjevic (pp. 1569-1579).

Secreted phospholipase B enzymes (PLB1) with high levels of N-linked glycosylation are proven fungal virulence determinants. We demonstrated that removal of N-linked glycans from secreted cryptococcal PLB1 leads to loss of enzyme activity. To determine if individual N-glycan attachment sites affect secretion of active enzyme, we altered three along the entire length of the protein, by site-directed mutagenesis, namely Asn56, Asn430 and Asn550 to Ala, in wild-type PLB1 (full length) and a glycosylphosphatidylinositol (GPI) anchorless version (PLB1^GPI−) that is hypersecreted due to lack of membrane association. Alteration of Asn56 and Asn550 in both PLB1 and PLB1^GPI− abolished enzyme secretion while alteration of Asn430 reduced secretion by 60%, following expression in Saccharomyces cerevisiae. Reduced secretion coincided with reduced enzyme in membranes and cell walls confirming a reduction in the rate of PLB1 transport to the cell surface. Deglycosylation of cryptococcal PLB1 increased its susceptibility to proteolysis suggesting that the absence of full glycosylation status leads to degradation of unstable PLB1, resulting in reduced traffic through the secretory pathway. We conclude that individual N-linked glycans are required for optimal transport of PLB1 to the cell surface and optimal secretion of both PLB1 and PLB1^GPI−.

Keywords: Phospholipase B; N-linked glycosylation; Secretion; GPI anchor; Cryptococcus neoformans; Site-directed mutagenesis

Cloning and expression of mouse cytosolic α-mannosidase (Man2c1) by Egidia Costanzi; Chiara Balducci; Renè Cacan; Sandrine Duvet; Aldo Orlacchio; Tommaso Beccari (pp. 1580-1586).

It is known that the neutral/cytosolic α-mannosidase (Man2c1) which can cleave α 1,2-, α 1,3-, and α 1,6-linked mannose residues, is stimulated by cobalt and is inhibited by furanose analogues swainsonine (SW) and 1,4-dideoxy-1,4-imino-d-mannitol (DIM). The enzyme is involved in the degradation of oligomannosides derived from dolichol intermediates and the degradation of newly synthesized glycoproteins. An immunological relationship has been demonstrated between the rat endoplasmic reticulum α-mannosidase and the cytosolic α-mannosidase. In fact antibodies raised against the soluble α-mannosidase recognized the membrane form of the ER α-mannosidase. A cDNA encoding the mouse cytosolic α-mannosidase was obtained by RZPD (Deutsches Ressourcenzentrum fur Genomforschung GmbH), Berlin, Germany. Comparison of the mouse genomic sequence with the cDNA sequence revealed the presence of 25 introns within the cytosolic α-mannosidase gene. The gene spans 11.5 kb from the major transcription initiation site to the RNA cleavage site. The transcription initiation site of mouse cytosolic α-mannosidase was mapped to 170 bases upstream of the ATG codon using 5′ RACE. Northern blotting analysis revealed expression of a major transcript of 3.8 kb in all tissues examined. COS cells transfected with the cDNA showed a 20-fold increase in cytosolic α-mannosidase activity. This enzyme activity was stimulated by cobalt and inhibited by DIM and EDTA. Furthermore we demonstrated that the expressed enzyme was active towards the radiolabeled substrate Man9GlcNAc1 giving the final product Man5GlcNAc1 through the formation of Man8GlcNAc1 isomer C as intermediate.

Keywords: Abbreviations; RACE; rapid amplification of cDNA ends; bp; base pair; kb; kilobase or 1000 bp; DMM; deoxymannojirimycin; KIF; kifunensine; HPLC; high-performance liquid chromatography; SW; swainsonine; ER; endoplasmic reticulum; DIM; 1,4-dideoxy-1,4-imino-; d; -mannitol; UTR; untraslated region; OSGn1; oligosaccharides possessing one GlNAc residue at the reducing end; PNGase; peptide: N-glycanaseCloning; Cytosolic α-mannosidase; Glycosidase; Man2c1; 5′ RACE

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BBA - General Subjects (v.1760, #10)