BBA - General Subjects (v.1760, #12)

Oxidation of acetylacetone catalyzed by horseradish peroxidase in the absence of hydrogen peroxide by Ana Paula Rodrigues; Luiz Marcos da Fonseca; Olga M. de Faria Oliveira; Iguatemy Lourenço Brunetti; Valdecir Farias Ximenes (1755-1761).
Horseradish peroxidase (HRP) is a plant enzyme widely used in biotechnology, including antibody-directed enzyme prodrug therapy (ADEPT). Here, we showed that HRP is able to catalyze the autoxidation of acetylacetone in the absence of hydrogen peroxide. This autoxidation led to generation of methylglyoxal and reactive oxygen species. The production of superoxide anion was evidenced by the effect of superoxide dismutase and by the generation of oxyperoxidase during the enzyme turnover. The HRP has a high specificity for acetylacetone, since the similar β-dicarbonyls dimedon and acetoacetate were not oxidized. As this enzyme prodrug combination was highly cytotoxic for neutrophils and only requires the presence of a non-human peroxidase and acetylacetone, it might immediately be applied to research on the ADEPT techniques. The acetylacetone could be a starting point for the design of new drugs applied in HRP-related ADEPT techniques.
Keywords: Horseradish peroxidase; Acetylacetone; Methylglyoxal; Reactive oxygen species; Antibody-directed enzyme prodrug therapy;

Biochemical characterization and molecular cloning of a plasminogen activator proteinase (LV-PA) from bushmaster snake venom by Eladio F. Sanchez; Liza F. Felicori; Carlos Chavez-Olortegui; Henrique B.P. Magalhaes; Ana L. Hermogenes; Marcelo V. Diniz; Inacio de L.M. Junqueira-de-Azevedo; Arinos Magalhaes; Michael Richardson (1762-1771).
The protein (LV-PA) from bushmaster (Lachesis muta muta) venom is a serine proteinase which specifically activates the inactive proenzyme plasminogen. LV-PA is a single chain glycoprotein with an apparent molecular mass of 33 kDa that fell to 28 kDa after treatment with N-Glycosidase F (PNGase F). Approximately 93% of its protein sequence was determined by automated Edman degradation of various fragments derived from a digestion with trypsin. A cDNA library of L. m. muta was constructed to generate expressed sequence tags (ESTs) and the plasminogen activator precursor cDNA was sequenced. The complete amino acid sequence of the enzyme was deduced from the cDNA sequence. LV-PA is composed of 234 residues and contains a single asparagine-linked glycosylation site, Asn-X-Ser, bearing sugars that account for ∼ 10% of the enzyme's total molecular mass of 33 kDa. The sequence of LV-PA is highly similar to the plasminogen activators (PAs) TSV-PA from Trimeresurus stejnegeri venom and Haly-PA from Agkistrodon halys. Furthermore, the mature protein sequence of LV-PA exhibits significant similarity with other viperidae venom serine proteinases which affect many steps of hemostasis, ranging from the blood coagulation cascade to platelet function. The Michaelis constant (Km) and the catalytic rate constant (kcat) of LV-PA on four chromogenic substrates were obtained from Lineweaver–Burk plots. In addition, we used an indirect enzyme-linked immunoabsorbent assay (ELISA) to explore the phylogenetic range of immunological cross-reactivity (using antibodies raised against LV-PA) with analogous serine proteinases from two viperidae venoms and mammals.
Keywords: Plasminogen activator; Fibrinolysis; cDNA; Bushmaster; Serine proteinase; Lachesis muta;

The effects of concentration and molecular weight of neutral dextrans on the adhesion of human red blood cells (RBC) to albumin-coated glass have been investigated using a parallel-plate flow chamber. Results indicate that the adhesion is markedly increased in the presence of 70 kDa and 500 kDa dextran, with this increase reflected by both the number of cells adhering and the strength of the adhesion. This increased adhesiveness is attributed to reduced surface concentrations of the large polymers and hence attractive forces due to depletion interaction. Depletion interaction brings the adjacent surfaces closer, leading to an increased number of binding sites available to the cell and thus more efficient and stronger adhesion of single cells. Our results suggest that depletion might play a role in other specific cell–cell or cell–surface interactions via initiating close contacts to allow specific binding.
Keywords: Adhesion; Depletion interaction; Dextran; Red blood cell;

Carboxylated chitooligosaccharides (CCOS) inhibit MMP-9 expression in human fibrosarcoma cells via down-regulation of AP-1 by Niranjan Rajapakse; Moon-Moo Kim; Eresha Mendis; Ronghua Huang; Se-Kwon Kim (1780-1788).
Matrix metalloproteinases (MMPs) play a fundamental role in invasion and metastasis of tumor and, recent advances in medicinal chemistry have approached designing of MMP inhibitors with desired structural properties, selectivity and bioavailability. In the present study, novel low-molecular-weight carboxylated chitooligosaccharides (CCOS) were evaluated for their MMP-9 inhibitory effect on human fibrosarcoma cell line (HT1080). In zymography experiments, a clear dose-dependent inhibition on MMP-9 mediated gelatinolytic activities were observed in HT1080 cells following treatment with CCOS. Further, transfection studies carried out with MMP-9 and AP-1 reporter constructs suggested that the observed reduction in MMP-9 expression was due to down-regulation of MMP-9 transcription that mediated via inhibition of AP-1. Moreover, expression of c-Fos protein levels in cytoplasm and nucleus confirmed that CCOS could inhibit AP-1 expression but not its translocation. However, in the presence of CCOS, NF-κB and TIMP-1 expression levels remained constant. More importantly, inhibition of MMP-9 expression clearly led to inhibit tumor invasiveness that was studied with reconstituted basement membrane matrix proteins coated synthetic membranes. Taken together, this study discusses MMP-9 inhibition potential of CCOS and their involvement to demote degradation and cellular invasion of extracellular matrix (ECM) and basement membrane. Thus, control of MMP-9 expression by CCOS has considerable significance for the regulation of tumor progression.
Keywords: Matrix metalloproteinase-9 (MMP-9); Carboxylated chitooligosaccharide (CCOS); Activator protein-1 (AP-1); Transcriptional regulation;

Deletion of selenoprotein P upregulates urinary selenium excretion and depresses whole-body selenium content by Raymond F. Burk; Kristina E. Hill; Amy K. Motley; Lori M. Austin; Brooke K. Norsworthy (1789-1793).
Deletion of the mouse selenoprotein P gene (Sepp1) lowers selenium concentrations in many tissues. We examined selenium homeostasis in Sepp1 / and Sepp1 +/+ mice to assess the mechanism of this. The liver produces and exports selenoprotein P, which transports selenium to peripheral tissues, and urinary selenium metabolites, which regulate whole-body selenium. At intakes of selenium near the nutritional requirement, Sepp1 / mice had whole-body selenium concentrations 72 to 75% of Sepp1 +/+ mice. Genotype did not affect dietary intake of selenium. Sepp1 / mice excreted in their urine approximately 1.5 times more selenium in relation to their whole-body selenium than did Sepp1 +/+ mice. In addition, Sepp1 / mice gavaged with 75SeO3 2− excreted 1.7 to 2.4 times as much of the 75Se in the urine as did Sepp1 +/+ mice. These findings demonstrate that deletion of selenoprotein P raises urinary excretion of selenium. When urinary small-molecule 75Se was injected intravenously into mice, over 90% of the 75Se appeared in the urine within 24 h, regardless of selenium status. This shows that urinary selenium is dedicated to excretion and not to utilization by tissues. Our results indicate that deletion of selenoprotein P leads to increased urinary selenium excretion. We propose that the absence of selenoprotein P synthesis in the liver makes more selenium available for urinary metabolite synthesis, increasing loss of selenium from the organism and causing the decrease in whole-body selenium and some of the decreases observed in tissues of Sepp1 / mice.
Keywords: Selenium homeostasis; Selenoprotein P; Urinary selenium excretion;

Acridine group of dyes are well known in the field of development of probes for nucleic acid structure and conformational determination because of their relevance in the development of novel chemotherapeutic agents, footprinting agents and for gene manipulation in biotechnology and medicine. Here, we report the interaction of 9-N,N-dimethylaniline decahydroacridinedione (DMAADD), a new class of dye molecule with calf thymus DNA (CT-DNA) which has been studied extensively by means of traditional experimental and theoretical techniques. The changes in the base stacking of CT-DNA upon the binding of DMAADD are reflected in the circular dichroic (CD) spectral studies. Competitive binding study shows that the enhanced emission intensity of ethidium bromide (EB) in presence of DNA was quenched by the addition of DMAADD indicating that it displaces EB from its binding site in DNA and the apparent binding constant has been estimated to be (3.3 ± 0.2) × 105 M− 1. This competitive binding study and further fluorescence experiments reveal that DMAADD is a moderate binder of CT-DNA, while viscosity measurements show that the mode of binding is partial intercalation. Generally, one would expect increase in the melting temperature (T m) of DNA in presence of intercalators. Interestingly, an unusual decrease in melting temperature (ΔT m of − 4 ± 0.2 °C) of DNA by the addition of DMAADD was observed. From our knowledge such a decreasing trend in melting point was not reported before for all the possible modes of binding. Molecular modeling gave the pictorial view of the binding model which clearly shows that of the various mode of binding, the dye prefers the major groove binding to the sites rich in GC residues and to the sites rich in AT residues it prefers intercalation mode of binding either through major or minor groove with the inclusion of the N,N-dimethylaniline (DMA) group inside the double helix which has been stacked in between the bases, under physiological relevant pH of 7.5.
Keywords: Acridinedione; DNA binding; Dual binding behaviour; Modelling studies;

β-amyloid (Aβ) is the primary protein component of senile plaques in Alzheimer's disease and is believed to be associated with neurotoxicity in the disease. We and others have shown that Aβ binds with relatively high affinity to clustered sialic acid residues on cell surfaces and that removal of cell surface sialic acids attenuate Aβ toxicity. In the current work, we have prepared sialic acid conjugated dendrimeric polymers and assessed the ability of these sialic acid conjugated dendrimers to prevent Aβ toxicity. Flow cytometry was used to analyze viability of SH-SY5Y neuroblastoma cells and the effects of soluble and clustered sialic acid mimics on Aβ cell toxicity. Soluble sialic acid attenuation of Aβ induced toxicity was effective only at high sialic acid concentrations and low Aβ concentration. The sialic acid conjugated dendrimeric polymers were able to attenuate Aβ toxicity at micromolar concentrations, or approximately three orders of magnitude lower concentrations than the soluble sialic acid. The toxicity prevention properties of the sialic acid modified dendrimers were a function of dendrimer size. This work may lead to the development of new classes of therapeutics for the prevention of Aβ toxicity.
Keywords: Alzheimer's disease; Amyloid; Dendrimer; Sialic acid; Toxicity;

Double roles of hydroxycinnamic acid derivatives in protection against lysozyme oxidation by Hongping Zhu; Shimou Chen; Shumei Hao; Zhaoxia Zhang; Wenfeng Wang; Side Yao (1810-1818).
Oxidative damage to protein has been implicated in a number of diseases. Much interest has been focused on preventing oxidative damage to protein. Here we showed that hydroxycinnamic acid derivatives (HCA) were able to inhibit the cross-linking of protein induced by riboflavin-mediated photooxidation. HCA were also found to strongly protect lysozyme from gamma rays irradiation. The antioxidative properties of HCA were further studied by laser flash photolysis. Mechanism of antioxidant activities of HCA on lysozyme oxidation was discussed. HCA were found to protect protein against oxidation by scavenging oxidizing species and repairing the damaged protein.
Keywords: Hydroxycinnamic acid derivatives; Protein oxidation; Antioxidant; Protection; Mechanism;

A genomic region from the thermophilic, filamentous, nondiazotrophic cyanobacterium Phormidium laminosum including nrtC and nrtD was cloned and sequenced. These genes encode NrtC and NrtD, the ATP-binding subunits of the ABC bispecific transporter of nitrate/nitrite NRT. We report a different nrtC sequence from the one previously reported (Merchán et al., Plant Mol. Biol. 28:759–766, 1995) and we identified the presence of nrtD gene downstream nrtC in the nirA operon. Each gene was expressed in E. coli cells as a hexahistidine-tagged fusion protein. The recombinant proteins (His6NrtC and His6NrtD) were purified, and their ability to catalyze the hydrolysis of ATP and other nucleosides triphosphate was characterized. Both subunits showed its maximum ATPase activity at 45–50 °C and pH 8.0, and similar K m (0.49 and 0.43 mM) and V max (0.085 and 0.114 U mg− 1 protein, respectively) values were calculated. The native NrtC subunit purified from nitrogen-starved cells of P. laminosum also hydrolyzed ATP in vitro in the absence of other components of NRT. These findings indicated that NrtC and NrtD are responsible for ATP-hydrolysis to energize the active transporter NRT. The effect of some activators (Mg2+) and inhibitors (ADP) on the ATPase activity of the subunits was assessed as well as the effect of some potential regulatory metabolites on His6NrtC. The existence in vitro of homodimers of either NrtC or NrtD but not heterodimers of both subunits was confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry and/or electrophoresis in non-denaturing conditions. Finally, the existence in vivo of NrtC-NrtD heterodimers is discussed.
Keywords: NrtC; NrtD; Nitrate/nitrite transport; ATP hydrolysis; Cyanobacteria; Phormidium laminosum;

Electrochemical analysis of the effect of Ca2+ on cardiolipin–cytochrome c interaction by Yinxi Huang; Lei Liu; Chang Shi; Junyi Huang; Genxi Li (1827-1830).
Mitochondrial Ca2+ has been considered a trigger for the release of cytochrome c, which is a critical and early event in the induction of cell apoptosis, although the molecular mechanism underlying this effect is still not fully understood. Here we investigate the interaction between cytochrome c and cardiolipin and the effect of Ca2+ on this interaction using electrochemical methods. Experimental results revealed that modification of cardiolipin onto the surface of a pyrolytic graphite electrode could lead to a rapid direct electron transfer of cytochrome c through the electrostatic interaction between the protein and the cardiolipin. Addition of Ca2+ to the test solution containing cytochrome c could cause the decrease of the redox peaks of the protein, and the peaks could be recovered when Ca2+ was chelated by ethylenediaminetetraacetate. The cardiolipin–cytochrome c interaction and the Ca2+ effect were also investigated with the variation of the charges of lipids, buffer solutions, reaction time, and valencies of cations for comparison.
Keywords: Cytochrome c; Cardiolipin; Calcium; Molecular interaction;

Is the expression of kinin B1 receptor related to intrahepatic vascular response? by Marcia R. Nagaoka; Luciana Gomiero; Felipe O. Teixeira; Fernando G. Agostino; José Eduardo P. Pouza; Priscila Mimary; Durval R. Borges; Maria Kouyoumdjian (1831-1836).
Bradykinin elicits an intrahepatic vascular response (IHVR) mediated by the constitutive B2 receptor (B2R). The biological effects of kinins may also be mediated by the inducible B1 receptor (B1R). Aim: To verify if the hepatic B1R expression modulates IHVR to kinins. Method: We evaluated the ability of bradykinin and B1R agonists to elicit an IHVR in normal rats and in those submitted to acute or chronic inflammatory stimuli, fibrosis, cirrhosis, or hepatic regeneration. Results: Bradykinin-induced IHVR was similar in all groups. B1R agonists did not elicit in any of them either a hypertensive or a hypotensive response. B1 receptor induction was observed in all experimental groups (Western blot), except for the acute inflammatory group. Conclusion: B1R hepatic expression did not modulate IHVR to kinins.
Keywords: Bradykinin; Des-Arg9-bradykinin; B1 receptor; Portal hypertension; Liver fibrosis;

Expression of aggrecan(ases) during murine preadipocyte differentiation and adipose tissue development by Gabor Voros; John D. Sandy; Désiré Collen; H. Roger Lijnen (1837-1844).
The expression and potential functional role of aggrecan in adipogenesis and adipose tissue development was investigated in murine models of obesity. Aggrecan, as well as the two aggrecanases ADAMTS-4 and ADAMTS-5 (A Disintegrin And Metalloproteinase with Thrombospondin motif) mRNAs, are expressed in subcutaneous (SC) and gonadal (GON) adipose tissues of mice. Their presence was confirmed by western blotting using adipose tissue extracts. In mice with nutritionally induced obesity (high fat diet) as well as in lean controls, aggrecan mRNA expression was downregulated whereas ADAMTS-4 and ADAMTS-5 were upregulated with time. In mice with genetically determined obesity (ob/ob), ADAMTS-5 mRNA was upregulated in both SC and GON adipose tissues, as compared to wild-type (WT) mice (p  < 0.001). Enhanced aggrecanase expression levels in these tissues were associated with significantly elevated levels of G1-NITEGE, a degradation product of aggrecan. Thus, aggrecan levels were high at the early stages of adipose tissue development in mice, whereas its production decreased and its degradation increased during development of obesity. A functional role of aggrecan in promoting early stages of adipogenesis is supported by the findings that it stimulated the in vitro differentiation of 3T3-F442A preadipocytes and the de novo in vivo accumulation of fat in Matrigel plaques injected into WT mice. Proteoglycans in the extracellular matrix of adipose tissue, such as aggrecan, may contribute to the regulation of lipid uptake and obesity in mice.
Keywords: Aggrecan; ADAMTS; Obesity; Adipogenesis;

The present article reports a low molecular weight aspartic protease inhibitor, API, from a newly isolated thermo-tolerant Bacillus licheniformis. The inhibitor was purified to homogeneity as shown by rp-HPLC and SDS-PAGE. API is found to be stable over a broad pH range of 2–11 and at temperature 90 °C for 2 1/2 h. It has a Mr (relative molecular mass) of 1363 Da as shown by MALDI-TOF spectra and 1358 Da as analyzed by SDS-PAGE .The amino acid analysis of the peptide shows the presence of 12 amino acid residues having Mr of 1425 Da. The secondary structure of API as analyzed by the CD spectra showed 7% α-helix, 49% β-sheet and 44% aperiodic structure. The Kinetic studies of Pepsin–API interactions reveal that API is a slow-tight binding competitive inhibitor with the IC50 and K i values 4.0 nM and (3.83 nM–5.31 nM) respectively. The overall inhibition constant K i⁎ value is 0.107 ± 0.015 nM. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E +  I  ⇄ (k 4, k 5) EI ⇄ (k 6, k 7) EI⁎. Rate constant k 6  = 2.73 ± 0.32 s− 1 reveals a fast isomerization of enzyme–inhibitor complex and very slow dissociation as proved by k 7  = 0.068 ± 0.009 s− 1. The Rate constants from the intrinsic tryptophanyl fluorescence data is in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI⁎.
Keywords: Aspartic protease inhibitor; Pepsin; Bacillus licheniformis; Enzyme kinetic; Slow-tight binding inhibition;

Saccharomyces cerevisiae Atm1p has been cloned, over-expressed and purified from a yeast expression system. The sequence includes both the soluble ATPase and transmembrane-spanning domains. With the introduction of an N-terminal Kozak sequence and a C-terminal (His)6-tag, a yield of 1 mg of Atm1p was obtained from 3 g wet yeast cells, which is comparable to other membrane-associated proteins isolated from eukaryotic expression systems. The ATPase activity of Atm1p is sensitive to sodium vanadate, a P-type ATPase inhibitor, with an IC50 of 4 μM. MgADP is a product inhibitor for Atm1p with an IC50 of 0.9 mM. The Michaelis–Menten constants V max, K M and k cat of Atm1p were measured as 8.7 ± 0.3 μM/min, 107 ± 16 μM and 1.24 ± 0.06 min− 1, respectively. A plot of ATPase activity versus concentration of Atm1p exhibits a nonlinear relationship, suggesting an allosteric response and an important role for the transmembrane domain in mediating both ATP hydrolysis and MgADP release. The metal dependence of Atm1p ATPase activity demonstrated a reactivity order of Mg2+  > Mn2+  > Co2+, while each divalent ion was found to be inhibitory at higher concentrations. The activation and inhibitory effect of phospholipids suggest that formation of a lipid–micelle complex is important for enzymatic activity and stability. Structural analysis of Atm1p by CD spectroscopy suggested a similarity of secondary structure to that found for other members of this ABC protein family.
Keywords: Atm1p; ABC7 transporter; Assay; Vesicles; Conformational gating; ATPase activity;

Down-regulation of ABCB1 transporter by atorvastatin in a human hepatoma cell line and in human peripheral blood mononuclear cells by Alice Cristina Rodrigues; Rui Curi; Luiz R.G. Britto; Ivanise M.M. Rebbechi; Mario H. Hirata; Marcelo C. Bertolami; Marcia M.S. Bernik; Egidio L. Dorea; Rosario D.C. Hirata (1866-1873).
Purpose. The effect of atorvastatin, an HMG-CoA reductase inhibitor, on expression and activity of the drug transporter ABCB1 in HepG2 cells and peripheral blood mononuclear cells (PBMCs) was examined. Methods. Localization and expression of ABCB1 in hepatocytes was examined by indirect immunofluorescence. Expression of ABCB1 mRNA and ABCB1 activity were examined in atorvastatin-treated and control cells and PBMCs using real-time PCR and Rhodamine 123 efflux assay. Results. Immunohistochemical analysis revealed that ABCB1 is located at the apical membrane of the bile canaliculi. Atorvastatin at 10 and 20 μM up-regulated ABCB1 expression resulting in a significant 1.4-fold increase of the protein levels. Treatment of HepG2 cells with 20 μM atorvastatin caused a 60% reduction on mRNA expression (p  < 0.05) and a 41% decrease in ABCB1-mediated efflux of Rhodamine123 (p  < 0.01) by flow cytometry. Correlation was found between ABCB1 mRNA levels and creatine kinase (r  = 0.30; p  = 0.014) and total cholesterol (r  = − 0.31; p  = 0.010). Conclusions. Atorvastatin leads to decreased ABCB1 function and modulates ABCB1 synthesis in HepG2 cells and in PBMCs. ABCB1 plays a role in cellular protection as well as in secretion and/or disposition, therefore, inhibition of ABCB1 synthesis may increase the atorvastatin efficacy, leading to a more pronounced reduction of plasma cholesterol.
Keywords: ABCB1; Atorvastatin; HepG2; ABCB1 mRNA; PBMC;

Mutation of Lys242 allows Δ32-enoyl-CoA isomerase to acquire enoyl-CoA hydratase activity by Wenhua Yu; Xiusheng Chu; Guisheng Deng; Xiaojun Liu; Gong Chen; Ding Li (1874-1883).
We report here a novel example of generating hydratase activity through site-directed mutagenesis of a single residue Lys242 of rat liver mitochondrial Δ32-enoyl-CoA isomerase, which is one of the key enzymes involved in fatty acid oxidation and a member of the crotonase superfamily. Lys242 is at the C-terminal of the enzyme, which is far from the active site in the crotonase superfamily and forms a salt bridge with Asp149. A variety of mutant expression plasmids were constructed, and it was observed that mutation of Lys242 to nonbasic residues allowed the mutants to have enoyl-CoA hydratase activity confirmed by HPLC analysis of the incubation mixture. Kinetic studies of these mutants were carried out for both isomerase and hydratase activities. Mutant K242C showed a k cat value of 1.0 s− 1 for hydration reaction. This activity constitutes about 10% of the total enzyme activity, and the remaining 90% is its natural isomerase activity. To the best of our knowledge, this is the first report on the generation of functional promiscuity through single amino acid mutation far from the active site. This may be a simple and efficient approach to designing a new enzyme based on an existing template. It could perhaps become a general methodology for facilitating an enzyme to acquire a type enzymatic activity that belongs to another member of the same superfamily, by interrupting a key structural element in order to introduce ambiguity, using site-directed mutagenesis.
Keywords: Enoyl-CoA isomerase; Enoyl-CoA hydratase; Crotonase superfamily; Structural alignment; Salt bridge; Inter-subunit interaction; MCPF-CoA; 2-octynoyl-CoA;

The VP8 subunit protein of human rotavirus (HRV) plays an important role in viral infectivity and neutralization. Recombinant peptide antigens displaying the amino acid sequence M1ASLIYRQLL10, a linear neutralization epitope on the VP8 protein, were constructed and examined for their ability to generate anti-peptide antibodies and HRV-neutralizing antibodies in BALB/c mice. Peptide antigen constructs were expressed in E. coli as fusion proteins with thioredoxin and a universal tetanus toxin T-cell epitope (P2), in order to enhance the anti-peptide immune response. The peptide antigen containing three tandem copies of the VP8 epitope induced significantly higher levels of anti-peptide antibody than only a single copy of the epitope, or the peptide co-administered with the carrier protein and T-cell epitope. Furthermore, the peptide antigen containing three copies of the peptide produced significantly higher virus-neutralization titres, higher than VP8, indicating that a peptide antigen displaying repeating copies of the amino acid region 1–10 of VP8 is a more potent inducer of HRV-neutralizing antibodies than VP8 alone, and may be useful for the production of specific neutralizing antibodies for passive immunotherapy of HRV infection.
Keywords: Human rotavirus; VP8; Neutralization epitope; Peptide antigen; B- and T-cell epitope; Antibody response;

Involvement of caspase activation and mitochondrial stress in taxol-induced apoptosis of Epstein–Barr virus-infected Akata cells by Young-Ok Son; Ki-Choon Choi; Jeong-Chae Lee; Sung-Ho Kook; Suk-Kyeong Lee; Kenzo Takada; Yong-Suk Jang (1894-1902).
Taxol (paclitaxel) is one of the most potent antimicrotubule agents currently used in cancer chemoprevention and treatment. However, the effects of taxol on the induction of apoptosis in Epstein–Barr virus (EBV)-infected cells are unknown. This study investigated the mechanisms of taxol on cell cycle arrest and apoptosis induction using the EBV-infected cell line, Akata. Taxol treatment sensitively and dose-independently induced growth inhibition, cytotoxicity, and apoptosis in the cells, which was demonstrated by the decreased level of tritium incorporation and cell viability, the increased number of positively stained cells in the trypan blue staining and TUNEL assay, the increased population of cells in the sub-G0/G1 phase in flow cytometric analysis, and ladder formation of the genomic DNA. Treatment with z-VAD-fmk almost completely protected the cells from taxol-induced apoptosis indicating that the taxol-induced apoptosis of Akata cells is caspase-dependent. In addition, taxol-induced apoptosis is proposed to be associated with a lower mitochondrial membrane potential and G2/M arrest. However, the tubulin expression level doses not appear to be a direct mediator of taxol-induced apoptosis in cells. The presence of EBV in these cells was not related to the sensitivity of the cells to the induction of apoptosis by taxol. Overall, these results demonstrate that taxol induces apoptosis in EBV-infected Akata cells in a dose-independent manner, and that caspase activation and mitochondrial stress are involved in the induction.
Keywords: Taxol; EBV-infected Akata cell; Cell cycle arrest; Caspase activation; Apoptosis;

An ascorbate-reducible cytochrome b561 is localized in macrophage lysosomes by De-liang Zhang; Dan Su; Alajos Bérczi; Amy Vargas; Han Asard (1903-1913).
Cytochromes b561 (Cyts b561) are a family of intrinsic membrane proteins involved in ascorbate-mediated transmembrane electron transport. The chromaffin granule Cyt b561 (CGCytb) is believed to transport electrons donated by extravesicular ascorbate (ASC) across the membrane to intravesicular monodehydroascorbate (MDA) supporting catecholamine synthesis in neuroendocrine tissues. Another isoform, the duodenal Cyt b561 (Dcytb), was reported to have ferric reductase activity, possibly facilitating intestinal iron uptake. Herein, a new Cyt b561 homologue, LCytb (for lysosomal Cytb561) was found expressed in the late endosomal–lysosomal membrane. LCytb shared high sequence similarity with CGCytb (45% identity) and Dcytb (42% identity). Moreover, four heme-coordinating His residues, and putative ASC and MDA binding sites were highly conserved. Recombinant LCytb exhibited an ASC-reducible b-type Cyt absorbance spectrum with α-band maximum at 561 nm in the spectrum of the reduced protein. Northern blots and Western blots revealed that LCytb was predominantly expressed in lung, spleen, thymus, testis and placenta. In situ hybridization and immunofluorescence studies further demonstrated that the protein was expressed in the alveolar macrophages of the lung, in the white pulp of the spleen, widespread in the thymus, and in the Sertoli cells of the testis. Sequence analysis indicated the presence of a (DE)XXXL(LI)-type signal in the C-terminal of the protein, predicting a late endosomal–lysosomal subcellular localization. This localization was confirmed by double labeling experiments in RAW264.7 and 293 cells, stably transfected with LCytb.
Keywords: Lysosomal cytochrome b561 (LCytb); Chromaffin granule cytochrome b561 (CGCytb); Duodenal cytochrome b561 (Dcytb); Macrophage, Lysosome;

From fresh fruiting bodies of the wild ascomycete mushroom (Xylaria hypoxylon) a lectin with N-terminal sequence resemblance to a part of Aspergillus oryzae genome and only slight similarity to fungal immunomodulatory protein from the mushroom Flammulina velutipes was isolated. The protocol comprised extraction with water, precipitation from the aqueous extract using 80% saturated (NH4)2SO4, ion exchange chromatography on DEAE-cellulose and CM-cellulose, and then gel filtration by fast protein liquid chromatography on Superdex 75. Lectin activity was adsorbed on DEAE-cellulose and unadsorbed on CM-cellulose. The lectin appeared as a single band with a molecular mass of 14.4 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single 28.8-kDa peak in gel filtration on Superdex 75. The lectin exhibited highly potent antiproliferative activity toward tumor cell lines, and exerted a potent anti-mitogenic action on mouse splenocytes. The hemagglutinating activity of the lectin was inhibited by inulin and xylose. It was stable up to 35 °C. At 40 °C its hemagglutinating activity was reduced by 50%, and it dwindled to 12.5% of the original activity at 50 °C. The hemagglutinating activity was also sensitive to NaOH and HCl solutions. The hemagglutinating activity was unaffected by CaCl2 and ZnCl2, and was potentiated substantially in the presence of AlCl3 and FeCl3. The distinctive features of this lectin comprise a unique sugar specificity, and highly potent hemagglutinating, antiproliferative and anti-mitogenic activities. X. hypoxylon lectin differs in molecular mass, N-terminal sequence and sugar specificity from previously reported ascomycete mushroom lectins.
Keywords: Lectin; Mushroom; Xylaria hypoxylon; Purification;