Interaction of ceruloplasmin and 5-lipoxygenase

The interaction between ceruloplasmin (CP), the multicopper oxidase of human plasma, and 5-lipoxygenase (5-LO), the key enzyme of leukotriene synthesis, is shown for the first time. By Western-blotting and mass spectrometry of tryptic fragments, it is shown that 5-LO from protein extract of human leukocytes binds with immobilized CP. Dose-dependent influence of intact CP on leukotrienes synthesis is found: CP reduced leukotrienes synthesis in leukocytes in a dose above 50 μg/ml (normal CP concentration in plasma is about 300–400 μg/ml). Proteolyzed CP and apo-form of CP is unable to inhibit activity of 5-LO. CP increased activity of 5-LO at low doses (5–10 μg/ml). On the whole, the influence of CP on phagocytosis index of leukocytes coordinates with influence on activity of 5-LO: the index increased in the range of 2–10 μg/ml CP and decreased at doses of CP above 40 μg/ml. The dual role of CP in regulation of cellular response of leukocytes is discussed.     

Design and characterization of a maltose binding protein-linked growth factor for matrix engineering

The maltose-binding protein (MBP), which possesses a large number of exposed hydrophobic zones, can be used as a link for the immobilization of growth factors. The amount of immobilized MBP-vascular endothelial growth factors (VEGFs) for polystyrene surface was increased with respect to increasing protein, showing 1019 ng/cm² at 100 μg protein/ml. The phosphorylation of VEGF receptors in the MBP-VEGF stimulated HEK293/KDR cells as depicted from western blot analysis. Cell adhesion to a MBP-VEGF immobilized surface was mediated by the VEGF-VEGFR interaction. These results suggest that MBP-VEGFs are active and a MBP immobilization system can then anchor various bioactive proteins to hydrophobic surfaces.     

Reversible immobilization of uricase on conductive polyaniline brushes grafted on polyacrylonitrile film

Polyacrylonitrile film (PAN) surfaces were modified with chemical polymerization of conductive polyaniline (PANI) in the presence of potassium dichromate as an oxidizing agent. The conductive films were used for immobilization of uricase. The surface resistance of the conductive film in this work was found to be 0.97 kΩ/cm. The maximum amount of immobilized enzyme on conductive film containing 2.4% PANI was about 216 μg/cm². The optimum pH for free and immobilized enzymes was observed at 7.0 and 7.5, respectively. The K _m values for free and immobilized uricase were found to be 94 and 138 μM, respectively. V _max values were calculated as 1.87 and 1.63 U/mg protein for the free and immobilized enzymes, respectively. Immobilized uricase exhibited ~68% of its original activity even after 2 months of storage at 4 °C while the free enzyme lost its initial activity within 4 weeks.     

Impact of seropositivity to <Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> and anti-hHSP60 on cardiovascular events in hemodialysis patients

Autoimmunity to heat shock protein 60 (HSP60) has been related to atherosclerosis. Chlamydia pneumoniae (CP), the most studied infectious agent implicated in promoting atherosclerosis, produces a form of HSP60, which can induce an autoimmune response, due to high antigenic homology with human HSP60 (hHSP60). In this study, we evaluated the correlations among anti-hHSP60 antibodies, CP infection, and cardiovascular disease (CVD) in a high-risk population, such as patients undergoing hemodialysis (HD). Thirty-two patients (67.9 ± 13.9 years; male/female, 23:9) on regular HD were enrolled. Global absolute cardiovascular risk (GCR) was assessed using the Italian CUORE Project’s risk charts, which evaluate age, gender, smoking habits, diabetes, systolic blood pressure, and serum cholesterol. The occurrence of cardiovascular events during a 24-month follow-up was recorded. Seropositivity to CP and the presence of anti-hHSP60 antibodies were tested by specific enzyme-linked immunosorbent assays. Inflammation was assessed by measurement of C-reactive protein (CRP) serum levels. Fifteen healthy sex and age-matched (61.9 ± 9.5 years; male/female, 11:4) subjects were the control group. Fifteen of 32 patients resulted seropositive for CP. CP + patients were older than CP−, while they did not differ for GCR, CRP, and dialytic parameters. CVD incidence was significantly higher in CP+ (9 CP+ vs 2 CP−, p < 0.05). Cox analysis recognized that the incidence of CVD was independently correlated with seropositivity to CP (HR, 7.59; p = 0.01; 95% CI = 1.63–35.4). On the other hand, there were no significant differences in anti-hHSP60 levels among CP+, CP− and healthy subjects: 18.11 μg/mL (14.8–47.8), 31.4 μg/mL (23.2–75.3), and 24.72 μg/mL (17.7–41.1), respectively. Anti-hHSP60 did not correlate to GCR, CRP, and incidence of CVD. In conclusion, our data suggest that anti-hHSP60 autoimmune response is not related to CP infection and CP-related CVD risk in HD patients.     

Study of Interaction of Ceruloplasmin with Serprocidins

This paper describes formation of complexes of ceruloplasmin (CP) with such proteins of the serprocidin family as azurocidin (CAP37), neutrophilic elastase (NE), cathepsin G (CG), and proteinase 3 (PR3). We present evidence that serprocidins form complexes with CP at a molar ratio 1: 1. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, did not prevent the interaction of serprocidins with CP in the course of SDS-free disc electrophoresis. CP affected the activities of NE, CG, and PR3 as a competitive inhibitor with K _i ≈ 1 μM. Inhibitory effect of CP depended on ionic strength of the solution and was negligible at NaCl concentrations above 300 mM. In the mode of competitive inhibitors serprocidins suppressed oxidase activity of CP towards p-phenylenediamine. CAP37 displayed the strongest inhibitory effect (K _i ≈20 nM). Upon adding various serprocidins to human, rat, rabbit, dolphin, dog, horse, and mouse plasma only CAP37 would form a complex with CP. Synthetic peptide RKARPRQFPRRR (5–13, 61–63 CAP37) displaced CAP37 from its complex with CP. Adding CAP37 to the triple complex formed by CP, lactoferrin, and myeloperoxidase resulted in displacement of the latter from the complex. The dissociation constant of CAP37 with immobilized CP was 13 nM. Therefore, among serprocidins CAP37 can be regarded as the specific partner of CP.     

Preparation and characterization of epoxy-functionalized magnetic chitosan beads: laccase immobilized for degradation of reactive dyes

Cross-linked magnetic chitosan beads were prepared by phase-inversion technique in the presence of epichlorohydrin under alkaline condition, and used for covalent immobilization of laccase. The activity of the immobilized laccase on the magnetic chitosan was about 260 U (g/dry beads) with an enzyme loading of about 16.33 ± 0.39 mg [(g/dry beads) mg/g]. Kinetic parameters, V _max and K _m values were determined as 21.7 U/mg protein and 9.4 μM for free enzyme, and 15.6 U/mg protein and 19.7 μM for the immobilized laccase, respectively. The operational and thermal stabilities of the immobilized laccase were improved compared to free counterpart. The immobilized laccase was operated in a batch reactor for the decolorization of reactive dyes from aqueous solution. The laccase immobilized on magnetic chitosan beads was very effective for removal of textile dyes from aqueous solution which creates an important environmental problem in the discharged textile dying solutions.     

Practical applications of high-affinity, albumin-binding proteins from a group G streptococcal isolate

Binding proteins that have high affinities for mammalian plasma proteins that are expressed on the surface of bacteria have proven valuable for the purification and detection of several biologically important molecules from human and animal plasma or serum. In this study, we have isolated a high affinity albumin-binding molecule from a group G streptococcal isolate of bovine origin and have demonstrated that the isolated protein can be biotinylated without loss of binding activity and can be used as a tracer for quantification of human serum albumin (HSA). The binding protein can be immobilized and used as a selective capture reagent in a competitive ELISA format using a biotinylated HSA tracer. In this assay format, the sensitivity of detection for 50% inhibition of binding of HSA was less than 1 μg/ml. When attached to the bacterial surface, this binding protein can be used to deplete albumin from human plasma, as analyzed by surface-enhanced laser desorption ionization time of flight mass spectrometry.     

Myocardial energy metabolism in ischemic preconditioning and cardioplegia: A metabolic control analysis

For both, cardioplegia (CP) and ischemic preconditioning (IP), increased ischemic tolerance with reduction in infarct size is well documented. These cardioprotective effects are related to a limitation of high energy phosphate (HEP) depletion. As CP and IP have to be assumed to act by different mechanisms, their effects on myocardial HEP metabolism cannot be assumed to be identical. Therefore, a systematic analysis of myocardial HEP metabolism for both procedures and their combination was performed, addressing the question whether there are different effects on myocardial HEP metabolism by IP and CP. In this study, metabolic control analysis was used to analyze the regulation of HEP metabolism. In open chest pigs subjected to 45 min LAD occlusion (index ischemia), CP and IP preserved myocardial ATP (control (C) 0.14 ± 0.05 μmol/g wwt; CP: 0.95 ± 0.14, IP: 0.61 ± 0.12; p<0.05 C vs. CP and IP) and reduced myocardial necrosis (infarct size IA/RA: C: 90.0 ± 3.0%; CP: 0.0 ± 0.0% but patchy necroses; IP: 5.05 ± 2.1%; p<0.05 C vs. CP and IP). The effects on HEP metabolism, however, were different: CP acted predominantly by slowing down the breakdown of phosphocreatine (PCr) during early phases of ischemia (C: ΔPCr 0–2 min: 5.24 ± 0.32 μmol/g wwt; CP: ΔPCr 0–2 min: 3.38 ± 0.23 μmol/g wwt, p<0.05 vs. C), leaving ATP breakdown during later stages unaffected (C: ΔATP 5–45 min: 1.77 ± 0.11 μmol/g wwt CP: ΔATP 5–45 min: 1.59 ± 0.28 μmol/g wwt, n.s. vs. C). In contrast to CP, in IP PCr breakdown was even increased (IP: ΔPCr 0–2 min: 7.06 ± 0.34 μmol/g wwt, p<0.05 vs. C), but ATP depletion greatly attenuated (IP: ΔATP 5–45 min: 0.48 ± 0.10 μmol/g wwt, p<0.05 vs. C and CP). Combining IP and CP yielded an additive effect with slowing down the breakdown of both PCr (IP+CP: ΔPCr 0–2 min: 5.09± 0.35 μmol/g wwt, p<0.05 vs. C and IP) and ATP (IP+CP: ΔATP 5–45 min: 0.56 ± 0.48 μmol/g wwt, p<0.05 vs. C and CP), resulting in a higher ATP content at the end of index ischemia (1.86 ± 0.46 μmol/g wwt, p<0.05 vs. C, CP and IP). Compared to IP, combining IP+CP achieved also a further reduction in infarct size (IA/RA: 0.0 ± 0.0%, p<0.05 vs IP) and—compared to CP—a disappearance of the patchy necroses. {The concept of major differences in myocardial HEP metabolism during CP and IP is further supported at a molecular level by metabolic control analysis. CP but not IP slowed down the CK reaction velocity at high PCr levels. In contrast to CP exerting a continuous decline in vATPase for any given ATP level, in IP myocardium ATPase reaction velocity was even increased at higher ATP contents, whereas a marked decrease in ATPase reaction velocity was found if ATP levels decreased. The equilibrium of the CK-reaction remained unchanged following CP, whereas IP induced a changing CK equilibrium, which was the more shifted towards PCr the more myocardial HEP content decreased. The data demonstrate different effects of CP and IP on myocardial HEP metabolism, i.e. PCr and ATP breakdown as well as the apparent equilibrium of the creatine kinase (CK)-reaction. For these reasons the combination of the two protective interventions has an additive effect. (Mol Cell Biochem 278: 222–232, 2005)     

Changes in protein pattern during different developmental stages of somatic embryos in chickpea

Mature embryonic axes were used for chickpea (Cicer arietinum L.) regeneration via somatic embryogenesis. Qualitative and quantitative estimation of protein profile during somatic embryogenesis by SDS-PAGE and densitometric analysis showed differential expression of various storage proteins at different stages of somatic embryo development, which was compared with the profile of developing seeds. Total protein content in somatic embryos of chickpea increased from globular stage [2.9 μg mg⁻¹(f.m.)] to cotyledonary stage [4.8 μg mg⁻¹(f.m.)] and then started decreasing during onset of maturation and germination [up to 1.5 μg mg⁻¹(f.m.)]. Differential expression of seed storage proteins, late embryogenesis abundant (LEA) proteins and proteins related with stress response were documented at different stages of somatic embryogenesis. Germinating somatic embryos showed degradation products of several seed storage proteins and the appearance of new polypeptides (76.8, 67.6, 49.9 and 34.2 kDa), which were absent during differentiation of somatic embryos. A low molecular mass (17.7 kDa) polypeptide was uniformly present during all stages of somatic embryogenesis and it may belong to a group of stress-related proteins. This study describes the expression of true seed storage proteins like legumin, vicilin, convicilin and their subunits at different stages of somatic embryogenesis, which may serve as excellent markers for embryogenic pathway of regeneration in chickpea.     

生物素化ATP硫酸化酶的表达、固定化与应用

现代大规模焦测序技术的产生是DNA测序技术的一次革命,其关键技术之一是得到高活性的、固定于磁性微球表面的ATP硫酸化酶．生物素化的ATP硫酸化酶可以通过生物素与亲和素之间的特异结合特性固定在包被亲和素的磁性微球表面,但是利用化学修饰法将ATP硫酸化酶进行生物素化修饰很可能会影响酶的活性．利用融合表达策略,将大肠杆菌生物素酰基载体蛋白C端87个氨基酸肽段(BCCP87)与ATP硫酸化酶在大肠杆菌内融合表达,经SDS-PAGE和Western blot分析,表达的融合蛋白分子质量约为64 ku,并且能够在大肠杆菌内被生物素化．生物素化的ATP硫酸化酶能够与亲和素包被的磁珠结合,固定后的ATP硫酸化酶具有活性,并且能够用于定量检测焦磷酸盐(PPi)和焦测序,为今后建立高通量大规模焦测序系统提供了一个有效的工具酶．     

The turnover of cell surface proteins of carrot protoplasts

The covalent modification of cell surface proteins with N-hydroxysuccinimide esters of biotin was used to develop a strategy for following the turnover of proteins on the surface of carrot (Daucus carota L.) protoplasts. A biotinylation/internalisation assay was established which enabled the turnover of cell surface proteins to be examined by biochemical and immunocytochemical techniques. The detection of biotinylated proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that a variety of proteins on the surface of the protoplasts were covalently modified. Immunolocalisation of biotinylated proteins in protoplasts directly after their derivatisation, demonstrated that the proteins were initially restricted to the cell surface. Incubation of biotinylated protoplasts at 25 °C for 1 h resulted in the detection of biotin-labelled proteins on the cell surface and intracellularly. A small proportion of these proteins was associated with coated pits, the Golgi apparatus and vacuolar compartments. Biochemical analysis of internalised proteins revealed that a polypeptide of approximate M_r 100 000 was internalised by the protoplasts. Immunolabelling of a biotinylated protein of M_r 100 000 by an antibody raised against an isoform of a tobacco plasma-membrane H⁺-ATPase, strongly suggests that the plasma-membrane H⁺-ATPase is internalised by carrot protoplasts. The implications of these results are discussed within the context of endocytosis in plants. Received: 13 July 1998 / Accepted: 11 November 1998     

Analysis of 3D structural differences in the IgG-binding domains based on the interresidue average-distance statistics

It is well-known that the IgG-binding domain from staphylococcal protein A folds into a 3α helix bundle structure, while the IgG-binding domain of streptococcal protein G forms an (α + β) structure. Recently, He et al. (Biochemistry 44:14055–14061, 2005) made mutants of these proteins from the wild types of protein A and protein G strains. These mutants are referred to as protein A219 and protein G311, and it was showed that these two mutants have different 3D structures, i.e., the 3α helix bundle structure and the (α + β) structure, respectively, despite the high sequence identity (59%). The purpose of our study was to clarify how such 3D structural differences are coded in the sequences with high homology. To address this problem, we introduce a predicted contact map constructed based on the interresidue average-distance statistics for prediction of folding properties of a protein. We refer to this map as an average distance map (ADM). Furthermore, the statistics of interresidue distances can be converted to an effective interresidue potential. We calculated the contact frequency of each residue of a protein in random conformations with this effective interresidue potential, and then we obtained values similar to ϕ values. We refer to this contact frequency of each residue as a p(μ) value. The comparison of the p(μ) values to the ϕ values for a protein suggests that p(μ) values reveal the information on the folding initiation site. Using these techniques, we try to extract the information on the difference in the 3D structures of protein A219 and protein G311 coded in their amino acid sequences in the present work. The results show that the ADM analyses and the p(μ) value analyses predict the information of folding initiation sites, which can be used to detect the 3D difference in both proteins.     

Surface plasmon resonance imaging analysis of hexahistidine-tagged protein on the gold thin film coated with a calix crown derivative

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the hexahistidine-ubiquitin-tagged human parathyroid hormone fragment (His₆-Ub-hPTHF(1–34)) expressed inEscherichia coli. The hexahistidine-specific antibody was immobilized on a thin gold film coated with ProLinker^TM B, a novel calixcrown derivative with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrices. The soluble and insoluble fractions of anE. coli cell lysate were spotted onto the antibody-coated gold chip, which was then washed with buffer (pH 7.4) solution and dried. SPR imaging measurements were carried out to detect the expressed His₆-Ub-hPTHF (1–34). There was no discernible protein image in the uninduced cell lysate, indicating that non-specific binding of contaminant proteins did not occur on the gold chip surface. It is expected that the approach used here to detect affinity-tagged recombinant proteins using an SPR imaging technique could be used as a powerful tool for the analyses of a number of proteins in a high-throughput mode.     

Assessment of small ligand-protein interactions by electrophoretic mobility shift assay using DNA-modified ligand as a sensing probe

The interaction between a small ligand and a protein were assessed by electrophoretic mobility shift assay. A sensing probe was created by modifying the model ligand, biotin, with DNA. The complex of DNA-modified ligand and anti-biotin antibody or streptavidin as a target protein was analyzed by agarose gel electrophoresis. The band corresponding to the DNA-modified ligand was shifted in the presence of the target protein, and the intensities of the shifted bands were decreased by adding increasing concentrations of free ligand ranging from 0.1 μM to 100 μM. From this calibration the concentration of ligand in the samples could be determined, allowing for evaluation of the interaction between a small ligand and its target.     

Enhanced production and secretion of rutin and GABA in immobilized cells of mulberry tree (<Emphasis Type="Italic">Morus bombycis</Emphasis> K.)

Cell immobilization has been proposed as a useful technique for mass production and efficient purification of secondary metabolites. In this study, we compared the bio-productivity of ligand-free and Ca-alginate-immobilized mulberry cells for rutin and γ-amino butyric acid (GABA). In the leaves of Subong mulberry plants (M. bombycis K.) grown in a greenhouse, GABA accumulated as the leaves aged; a more than a 20-fold increase of GABA was observed in leaves undergoing senescence than in younger leaves. In contrast, more rutin was detected in mature leaves than in young leaves and those undergoing senescence. The production of total proteins in ligand-free leaf callus cells dramatically increased until 6 days after incubation in liquid suspension media (from 6.5 mg/g callus at day 0–14.5 mg/g callus), and by day 15 dropped to levels similar to those seen in the 0-day control. In contrast, immobilized cells showed a slight increase and then an insignificant decrease in protein content during the 15-day incubation period. Interestingly, immobilized mulberry cells more efficiently produced and secreted rutin and GABA into the suspension media than ligand-free cells. KN, a cytokinin, enhanced this production while 2,4-dichlorophenoxyacetic acid(2,4-D), an auxin, alleviated the effect of KN. As a result, incubation of the immobilized Subong cells in a full-strength Murashige and Skoog (MS) liquid medium containing 1 mg/l of 2,4-D and 0.1 mg/l KN, among the hormone combinations in the medium we tested, produced the highest amounts of rutin (8.2 μg/g callus cells) and GABA (305 μg/g callus cells) and secreted the largest amounts into the suspension media.     

A conidial protein (CP15) of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> contributes to the conidial tolerance of the entomopathogenic fungus to thermal and oxidative stresses

Aerial conidia are central dispersing structures for most fungi and represent the infectious propagule for entomopathogenic fungus Beauveria bassiana, thus the active ingredients of commercial mycoinsecticides. Although a number of formic-acid-extractable (FAE) cell wall proteins from conidia have been characterized, the functions of many such proteins remain obscure. We report that a conidial FAE protein, termed CP15, isolated from B. bassiana is related to fungal tolerance to thermal and oxidative stresses. The full-length genomic sequence of CP15 was shown to lack introns, encoding for a 131 amino acid protein (15.0 kDa) with no sequence identity to any known proteins in the NCBI database. The function of this new gene with two genomic copies was examined using the antisense-RNA method. Five transgenic strains displayed various degrees of silenced CP15 expression, resulting in significantly reduced conidial FAE protein profiles. The FAE protein contents of the strains were linearly correlated to the survival indices of their conidia when exposed to 30-min wet stress at 48°C (r ² = 0.93). Under prolonged 75-min heat stress, the median lethal times (LT₅₀s) of their conidia were significantly reduced by 13.6–29.5%. The CP15 silenced strains were also 20–50% less resistant to oxidative stress but were not affected with respect to UV-B or hyperosmotic stress. Our data indicate that discrete conidial proteins may mediate resistance to some abiotic stresses, and that manipulation of such proteins may be a viable approach to enhancing the environmental fitness of B. bassiana for more persisting control of insect pests in warmer climates.     

Artificial biofilm model – a useful tool for biofilm research

For biofilm studies, artificial models can be very helpful in studying processes in hydrogels of defined composition and structure. Two different types of artificial biofilm models were developed. Homogeneous agarose beads (50–500 μm diameter) and porous beads (260 μm mean diameter) containing pores with diameters from 10 to 80 μm (28 μm on average) allowed the embedding of cells, particles and typical biofilm matrix components such as proteins and polysaccharides. The characterisation of the matrix structures and of the distribution of microorganisms was performed by confocal laser scanning microscopy. The physiological condition of the embedded bacteria was examined by redox activity (CTC-assay) and membrane integrity (Molecular Probes LIVE/DEAD-Kit). Approximately 35% of the immobilised cells (Pseudomonas aeruginosa SG81) were damaged due to the elevated temperature required for the embedding process. It was shown that the surviving cells were able to multiply when provided with nutrients. In the case of homogeneous agarose beads, cell growth only occurred near the bead surface, while substrate limitation prevented growth of more deeply embedded cells. In the porous hydrogel, cell division was observed across the entire matrix due to better mass transport. It could be shown that embedding in the artificial gel matrix provided protection of immobilized cells against toxic substances such as sodium hypochlorite (0.5 mg/l, 30 min) in comparison to suspended cells, as observed in other immobilized systems. Thus, the model is suited to simulate important biofilm matrix properties. Received: 21 December 1999 / Received revision: 7 March 2000 / Accepted: 10 March 2000     

Determination of Optimal Protein Quantity Required to Identify Abundant and Less Abundant Soybean Seed Proteins by 2D-PAGE and MS

Optimizing the amounts of proteins required to separate and characterize both abundant and less abundant proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is critical for conducting proteomic research. In this study, we tested five different levels of soybean seed proteins (75, 100, 125, 150, and 200 μg) by 2D-PAGE. Following 2D-PAGE and spot excision, proteins were identified by mass spectrometry analysis. The number of visible protein spots was increased with an increase in the amount of protein loaded. The intensity of highly abundant proteins [β-conglycinin β-homotrimer and glycinin G4 (A5A4B3) precursors] increased linearly between 75 and 125 μg, whereas the proglycinin G3 (A1ab1b) homotrimer showed linearity between 75 and 150 μg. The spot intensity of less abundant proteins, glycinin G2 (A2b1a) precursor and proglycinin G3 (A1ab1b) homotrimer, increased linearly with an increase in the amount of protein through 200 μg, whereas spot intensity of β-conglycinin β-homotrimer and the allergen Gly m bd 28K increased linearly until 150 μg and did not increase further at 200 μg. These results suggest that 150 μg protein was a suitable amount for the separation of abundant proteins, and 200 μg protein was suitable for the separation of less abundant proteins prepared from soybean seeds. Mention of trade name, proprietary product or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or imply its approval to the exclusion of other products or vendors that also may be suitable.     

Anti-Bcl-2 family members,zfBcl-x<Subscript>L</Subscript> and zfMcl-1a,prevent cytochrome <Emphasis Type="Italic">c</Emphasis> release from cells undergoing betanodavirus-induced secondary necrotic cell death

Nervous necrosis virus (NNV)-induced, host cell apoptosis mediates secondary necrosis by an ill-understood process. In this study, redspotted grouper nervous necrosis virus (RGNNV) is shown to induce mitochondria-mediated necrotic cell death in GL-av cells (fish cells) via cytochrome c release, and anti-apoptotic proteins are shown to protect these cells from death. Western blots revealed that cytochrome c release coincided with disruption of mitochondrial ultrastructure and preceded necrosis, but did not correlate with caspases activation. To identify the mediator(s) of this necrotic process, a protein synthesis inhibitor (cycloheximide; CHX; 0.33 μg/ml) was used to block cytochrome c release as well as PS exposure and mitochondrial membrane permeability transition pore (MMP) loss. CHX (0.33 μg/ml) completely blocked viral protein B2 expression, and partly blocked protein A, protein α, and a pro-apoptotic death protein (Bad) expression. Overexpression of B2 gene increased necrotic-like cell death up to 30% at 48 h post-transfection, suggesting that newly synthesized protein (B2) may be involved in this necrotic process. Finally, necrotic death was prevented by overexpression of Bcl-2 family proteins, zfBcl-x_L and xfMcl-1a. Thus, new protein synthesis and release of cytochrome c are required for RGNNV-induced necrotic cell death, which can be blocked by anti-apoptotic Bcl-2 members. J.-L. Wu and J.-R. Hong contributed equally to the research.     

Application of an interferometric biosensor chip to biomonitoring an endocrine discruptor

RecombinantE. coli ACV 1003 (recA::lacZ) releasing β-galactosidase by a SOS regulon system, when exposed to DNA-damaging compounds, have been used to effectively monitor endocrine disruptors. Low enzyme activity of less than 10 units/mL, corresponding to a μg/L (ppb) range of an endocrine disruptor (tributyl tin, bisphenol A,etc.), can be rapidly determined, not by a conventional time-consuming and tedious enzyme assay, but by an alternative interferometric biosensor. Heavily boron-doped porous silicon for application as an interferometer, was fabricated by etching to form a Fabry-Perot fringe pattern, which caused a change in the refractive index of the medium including β-galactosidase. In order to enhance the immobilization of the porous silicon surface, a calyx crown derivative (ProLinker A) was applied, instead of a conventional biomolecular affinity method using biotin. This resulted in a denser linked formation. The change in the effective optical thickness versus β-galactosidase activity, showed a linear increase up to a concentration of 150 unit β-galactosidase/mL, unlike the sigmoidal increase pattern observed with the biotin.