BCA Method Research Articles

Galectin-3 Protein Regulates Mobility of N-cadherin and GM1 Ganglioside at Cell-Cell Junctions of Mammary Carcinoma Cells

Galectin-3 binding to cell surface glycoproteins, including branched N-glycans generated by N-acetylglucosaminyltransferase V (Mgat5) activity, forms a multivalent, heterogeneous, and dynamic lattice. This lattice has been shown to regulate integrin and receptor tyrosine kinase signaling promoting tumor cell migration. N-cadherin is a homotypic cell-cell adhesion receptor commonly overexpressed in tumor cells that contributes to cell motility. Here we show that galectin-3 and N-cadherin interact and colocalize with the lipid raft marker GM1 ganglioside in cell-cell junctions of mammary epithelial cancer cells. Disruption of the lattice by deletion of Mgat5, siRNA depletion of galectin-3, or competitive inhibition with lactose stabilizes cell-cell junctions. It also reduces, in a p120-catenin-dependent manner, the dynamic pool of junctional N-cadherin. Proteomic analysis of detergent-resistant membranes (DRMs) revealed that the galectin lattice opposes entry of many proteins into DRM rafts. N-cadherin and catenins are present in DRMs; however, their DRM distribution is not significantly affected by lattice disruption. Galectin lattice integrity increases the mobile fraction of the raft marker, GM1 ganglioside binding cholera toxin B subunit Ctb, at cell-cell contacts in a p120-catenin-independent manner, but does not affect the mobility of either Ctb-labeled GM1 or GFP-coupled N-cadherin in nonjunctional regions. Our results suggest that the galectin lattice independently enhances lateral molecular diffusion by direct interaction with specific glycoconjugates within the adherens junction. By promoting exchange between raft and non-raft microdomains as well as molecular dynamics within junction-specific raft microdomains, the lattice may enhance turnover of N-cadherin and other glycoconjugates that determine junctional stability and rates of cell migration.

Cell Adhesion-dependent Serine 85 Phosphorylation of Paxillin Modulates Focal Adhesion Formation and Haptotactic Migration via Association with the C-terminal Tail Domain of Talin

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration.

Determinação de Cu, Fe, Mn, Zn e do teor de proteína total em amostras de trigo e soja após procedimento de extração sequencial

A sequential extraction procedure was applied to wheat and soybean seed samples. The total protein content (determined by two distinct methods: Bradford and bicinchoninic acid-BCA) and distribution of Cu, Fe, Mn and Zn in each fraction was determined. The sequential extraction employed four different solutions: water, 0.5 mol L-1 NaCl, ethanol/water (70:30 v v-1) and 0.5 mol L-1 NaOH. For both samples, the highest concentration of metals was observed in those extracts associated with globulin-type proteins using NaCl solution. Regarding protein content, higher levels were obtained using the BCA method.

Taurine inhibits osteoblastic differentiation of aortic valve interstitial cells induced by beta-glycerophosphate, dexamethasone and ascorbic acid via the ERK pathway

PurposeAortic valve calcification (AVC) has been recognised as an active process characterised by osteoblastic differentiation of the aortic valve interstitial cells (AVICs). Taurine is a free β-amino acid and has...

Interleukin-6 (IL-6) Regulates Claudin-2 Expression and Tight Junction Permeability in Intestinal Epithelium

In inflammatory bowel diseases (IBD), intestinal barrier function is impaired as a result of deteriorations in epithelial tight junction (TJ) structure. IL-6, a pleiotropic cytokine, is elevated in IBD patients, although the role of IL-6 in barrier function remains unknown. We present evidence that IL-6 increases TJ permeability by stimulating the expression of channel-forming claudin-2, which is required for increased caudal-related homeobox (Cdx) 2 through the MEK/ERK and PI3K pathways in intestinal epithelial cells. IL-6 increases the cation-selective TJ permeability without any changes to uncharged dextran flux or cell viability in Caco-2 cells. IL-6 markedly induces claudin-2 expression, which is associated with increased TJ permeability. The colonic mucosa of mice injected with IL-6 also exhibits an increase in claudin-2 expression. The claudin-2 expression and TJ permeability induced by IL-6 are sensitive to the inhibition of gp130, MEK, and PI3K. Furthermore, expression of WT-MEK1 induces claudin-2 expression in Caco-2 cells. Claudin-2 promoter activity is increased by IL-6 in a MEK/ERK and PI3K-dependent manner, and deletion of Cdx binding sites in the promoter sequence results in a loss of IL-6-induced promoter activity. IL-6 increases Cdx2 protein expression, which is suppressed by the inhibition of MEK and PI3K. These observations may reveal an important mechanism by which IL-6 can undermine the integrity of the intestinal barrier.

Cyclin-dependent Kinase-5 Is a Key Molecule in Tumor Necrosis Factor-α-induced Insulin Resistance

The mechanism of TNF-α-induced insulin resistance has remained unresolved with evidence for down-regulation of insulin effector targets effects or blockade of proximal as well as distal insulin signaling events depending upon the dose, time, and cell type examined. To address this issue we examined the acute actions of TNF-α in differentiated 3T3L1 adipocytes. Acute (5-15 min) treatment with 20 ng/ml (~0.8 nm) TNF-α had no significant effect on IRS1-associated phosphatidylinositol 3-kinase. In contrast, TNF-α increased insulin-stimulated cyclin-dependent kinase-5 (CDK5) phosphorylation on tyrosine residue 15 through an Erk-dependent pathway and up-regulated the expression of the CDK5 regulator protein p35. In parallel, TNF-α stimulation also resulted in the phosphorylation and GTP loading of the Rho family GTP-binding protein, TC10α. TNF-α enhanced the depolymerization of cortical F-actin and inhibited insulin-stimulated glucose transporter-4 (GLUT4) translocation. Treatment with the MEK inhibitor, PD98059, blocked the TNF-α-induced increase in CDK5 phosphorylation and the depolymerization of cortical F-actin. Conversely, siRNA-mediated knockdown of CDK5 or treatment with the MEK inhibitor restored the impaired insulin-stimulated GLUT4 translocation induced by TNF-α. Furthermore, siRNA-mediated knockdown of p44/42 Erk also rescued the TNF-α inhibition of insulin-stimulated GLUT4 translocation. Together, these data demonstrate that TNF-α-mediated insulin resistance of glucose uptake can occur through a MEK/Erk-dependent activation of CDK5.

Differential Impact of Caveolae and Caveolin-1 Scaffolds on The Membrane Raft Proteome

Caveolae, a class of cholesterol-rich lipid rafts, are smooth invaginations of the plasma membrane whose formation in nonmuscle cells requires caveolin-1 (Cav1). The recent demonstration that Cav1-associated cavin proteins, in particular PTRF/cavin-1, are also required for caveolae formation supports a functional role for Cav1 independently of caveolae. In tumor cells deficient for Golgi β-1,6N-acetylglucosaminyltransferase V (Mgat5), reduced Cav1 expression is associated not with caveolae but with oligomerized Cav1 domains, or scaffolds, that functionally regulate receptor signaling and raft-dependent endocytosis. Using subdiffraction-limit microscopy, we show that Cav1 scaffolds are homogenous subdiffraction-limit sized structures whose size distribution differs from that of Cav1 in caveolae expressing cells. These cell lines displaying differing Cav1/caveolae phenotypes are effective tools for probing the structure and composition of caveolae. Using stable isotope labeling by amino acids in cell culture, we are able to quantitatively distinguish the composition of caveolae from the background of detergent-resistant membrane proteins and show that the presence of caveolae enriches the protein composition of detergent-resistant membrane, including the recruitment of multiple heterotrimeric G-protein subunits. These data were further supported by analysis of immuno-isolated Cav1 domains and of methyl-β-cyclodextrin-disrupted detergent-resistant membrane. Our data show that loss of caveolae results in a dramatic change to the membrane raft proteome and that this change is independent of Cav1 expression. The proteomics data, in combination with subdiffraction-limit microscopy, indicates that noncaveolar Cav1 domains, or scaffolds are structurally and functionally distinct from caveolae and differentially impact on the molecular composition of lipid rafts.

강우빈도해석에서 Bootstrap을 이용한 확률분포의 매개변수 추정에 대한 불확실성 해석

Bootstrap methods is the computer-based resampling method that estimates the standard errors and confidence intervals of summary statistics using the plug-in principle for assessing the accuracy or uncertainty of statistical estimates, and the BCa method among the Bootstrap methods is known much superior to other Bootstrap methods in respect of the standards of statistical validation. Therefore this study suggests the method of the representation and treatment of uncertainty in flood risk assessment and water resources planning from the construction and application of rainfall frequency analysis model considersing the uncertainty based on the nonparametric BCa method among the Bootstrap methods for the assessement of the estimation of probability rainfall and the effect of uncertainty considering the uncertainty of the parameter estimation of probability in the rainfall frequency analysis that is the most fundamental in flood risk assessement and water resources planning.

Activation of GluR6-containing Kainate Receptors Induces Ubiquitin-dependent Bcl-2 Degradation via Denitrosylation in the Rat Hippocampus after Kainate Treatment

We previously showed that Bcl-2 (B-cell lymphoma 2) is down-regulated in a kainate (KA)-induced rat epileptic seizure model. The underlying mechanism had remained largely unknown, but we here report for the first time that denitrosylation and ubiquitination are involved. Our results show that the S-nitrosylation levels of Bcl-2 are down-regulated after KA injection and that the GluR6 (glutamate receptor 6) antagonist NS102 can inhibit the denitrosylation of Bcl-2. Moreover, the ubiquitin-dependent degradation of Bcl-2 was found to be promoted after KA treatment, which could be suppressed by the proteasome inhibitor MG132 and the NO donors, sodium nitroprusside and S-nitrosoglutathione. In addition, experiments based on siRNA transfections were performed in the human SH-SY5Y neuroblastoma cell line to verify that the stability of Bcl-2 is causal to neuronal survival. At the same time, it was found that the exogenous NO donor GSNO could protect neurons when Bcl-2 is targeted. Subsequently, these mechanisms were morphologically validated by immunohistochemistry, cresyl violet staining, and in situ TUNEL staining to analyze the expression of Bcl-2 as well as the survival of CA1 and CA3/DG pyramidal neurons. NS102, GSNO, sodium nitroprusside, and MG132 contribute to the survival of CA1 and CA3/DG pyramidal neurons by attenuating Bcl-2 denitrosylation. Taken together, our data reveal that Bcl-2 ubiquitin-dependent degradation is induced by Bcl-2 denitrosylation during neuronal apoptosis after KA treatment.

Psychological stress has no association with salivary levels of β-defensin 2 and β-defensin 3

Recent studies suggest that stress can predispose an individual to the development of periodontal disease, but the exact biological mechanism is unknown. Considering that psychological stress can down-regulate the production of β-defensins (antimicrobial peptides produced in the oral cavity), the aim of the present study was to evaluate the association between stress and salivary levels of β-defensin 2 (HBD-2) and β-defensin 3 (HBD-3). For this purpose, seventy five volunteers, classified as periodontally healthy, were submitted to a psychological evaluation using a validated questionnaire (Questionnaire of Lipp-ISS). Following analysis of the questionnaires, the subjects were divided in two groups (Group A: Absence of stress and Group B: Presence of stress). Unstimulated saliva samples were collected and the concentration of total protein was determined using the BCA method, and the concentrations of HBD-2 and HBD-3 were determined by ELISA. The levels of total protein did not show a statistically significant difference between the groups. Analyses of HBD-2 and HBD-3 concentrations indicate that the stress condition was not associated with the levels of either peptide in saliva (P=0.3664 for HBD-2 and P=0.3608 for HBD-3). In periodontally healthy subjects, HBD-2 and HBD-3 levels are not influenced by stress.

Investigation of 99mTc-labelling of recombinant human interleukin-2 via hydrazinonicotinamide

Interleukin-2 (IL-2) when radiolabelled with (99m)Tc has been proved useful in imaging the side of lymphocytic infiltration in patients with autoimmune disorders and plays a significant role as a T-cell imaging agent. However, the labelling procedures used so far appeared to be rather complex and laborious. The aim of present study was to develop an efficient procedure of (99m)Tc-labelling of recombinant human interleukin-2 (rhIL-2) via hydrazinonicotinamide (HYNIC) to develop a dry kit formulation. Various molar ratios of rhIL-2/HYNIC (from 1:2 to 1:12) were used at the conjugation step. The conjugates were purified on a PD-10 column to remove the excess of unbound HYNIC, as well as of any aggregates. The final peptide concentration was quantified by the BCA method, and the number of HYNIC molecules incorporated into a rhIL-2 molecule was determined based on the reaction with 2-sulfobenzaldehyde. The (99m)Tc-labelling was optimized using various amounts of HYNIC-rhIL-2, (99m)Tc, SnCl(2), tricine and nicotinic acid (NA). Quality control included GF-HPLC, ITLC, SDS-PAGE and biological assay. Biodistribution studies were performed in Swiss mice and Wistar rats. Generally, the highest radiolabelling yields were achieved when the HYNIC-rhIL-2 conjugates of ca. 2-4 HYNIC molecule substitution ratios were used. The optimal pH of the reaction medium was found to be in the range of 6.5 to 7.0. GF-HPLC analysis indicated that monomer and aggregates of (99m)Tc-HYNIC-rhIL-2 are formed during radiolabelling. At optimized conditions of wet radiolabelling, the (99m)Tc-HYNIC-rhIL-2 monomer was obtained with radiochemical purity >99%, specific activity of ca. 4 GBq/mg rhIL-2 and overall yield of ca. 65%. The two-vial freeze-dried kit was prepared: the first vial contained 30 μg HYNIC-rhIL-2, co-ligands, buffer and antioxidant; the second vial contained tricine and SnCl(2). The monomer of (99m)Tc-HYNIC-rhIL-2 was obtained by gel chromatography on a PD-10 column. No differences between labelled and unlabelled IL2 in terms of biological activity were observed. Our study shows that rhIL-2 can be efficiently radiolabelled with (99m)Tc via HYNIC, with tricine and NA as co-ligands using a two-vial freeze-dried kit. This enables the preparation of sterile and ready-to-use (99m)Tc-HYNIC(tricine,NA)-rhIL-2 within 1 h.

Efficient Inhibition of Interfacial Nonspecific Interaction to Create Practically Utilizable High Ferritin-Response Immunolatex

Immunolatex particles (LAmP-s), which were prepared by covalently co-immobilizing antiferritin and a mixture of pentaethylenehexamine-ended poly(ethylene glycol) (N6-PEG) of two different chain lengths onto the surface of polystyrene submicroparticles, were formulated with various antiferritin loads. Following the quantification of the bound antiferritin, as well as the differentiation of the physically adsorbed antiferritin from chemically bound antiferritin, using the copper reduction/bicinchoninic acid reaction (the Micro BCA method), dynamic light scattering (DLS) and electrophoretic mobility (mu(e)) measurements were performed to characterize the size, homogeneity, and surface charge of the complex. Compared to the control immunolatex particles, which were prepared similarly but traditionally using bovine serum albumin (BSA) as a blocking agent (LAB-s), the LAmP-s complex showed a difference only in the surface charge property, because of the altered surface treatment in the case of the LAmP-s (PEGylation) and LAB-s complexes (BSA covering). However, the LAmP-s complex was much more reactive than the LAB-s complex, not only in phosphate buffer (PB, 10 mM, pH 7.4) but also in 100% fetal bovine serum (FBS), as measured by the turbidimetric monitoring method. The electrical repulsion between the negatively charged LAB-s complex and the antigen was the primary obstacle in the former case, and the overwhelming nonspecific deposition of contaminants from FBS onto the LAB-s complex was the main reason in the latter case. Moreover, the PEGylation procedure allowed the LAmP-s complex to possess invariable size and reactivity for at least one month at 4 degrees C without salt, and colloidal stability at high salt concentrations up to 2.0 M for at least one week, obviously demonstrating that the PEG surface modification technique described in this paper is a promising method of constructing efficient immunoassay systems.

Accelerated Fatty Acid Oxidation in Muscle Averts Fasting-induced Hepatic Steatosis in SJL/J Mice

The accumulation of triglycerides (TG) in the liver, designated hepatic steatosis, is characteristically associated with obesity and insulin resistance, but it can also develop after fasting. Here, we show that fasting-induced hepatic steatosis is under genetic control in inbred mice. After a 24-h fast, C57BL/6J mice and SJL/J mice both lost more than 20% of body weight and approximately 60% of total body TG. In C57BL/6J mice, TG accumulated in liver, producing frank steatosis. In striking contrast, SJL/J mice failed to accumulate any hepatic TG even though they lost nearly as much adipose tissue mass as the C57BL/6J mice. Mice from five other inbred strains developed fasting-induced steatosis like the C57BL/6J mice. Measurements of the uptake of free fatty acids (FA) in vivo and in vitro demonstrated that SJL/J mice were protected from steatosis because their heart and skeletal muscle took up and oxidized twice as much FA as compared with C57BL/6J mice. As a result of this muscle diversion, serum-free FA and ketone bodies rose much less after fasting in SJL/J mice as compared with C57BL/6J mice. When livers of SJL/J and C57BL/6J mice were perfused with similar concentrations of FA, the livers took up and esterified similar amounts. We conclude that SJL/J mice express one or more variant genes that lead to enhanced FA uptake and oxidation in muscle, thereby sparing the liver from FA overload in the fasting state.

Interaction of Mint2 with TrkA Is Involved in Regulation of Nerve Growth Factor-induced Neurite Outgrowth

TrkA receptor signaling is essential for nerve growth factor (NGF)-induced survival and differentiation of sensory neurons. To identify possible effectors or regulators of TrkA signaling, yeast two-hybrid screening was performed using the intracellular domain of TrkA as bait. We identified muc18-1-interacting protein 2 (Mint2) as a novel TrkA-binding protein and found that the phosphotyrosine binding domain of Mint2 interacted with TrkA in a phosphorylation- and ligand-independent fashion. Coimmunoprecipitation assays showed that endogenous TrkA interacted with Mint2 in rat tissue homogenates, and immunohistochemical evidence revealed that Mint2 and TrkA colocalized in rat dorsal root ganglion neurons. Furthermore, Mint2 overexpression inhibited NGF-induced neurite outgrowth in both PC12 and cultured dorsal root ganglion neurons, whereas inhibition of Mint2 expression by RNA interference facilitated NGF-induced neurite outgrowth. Moreover, Mint2 was found to promote the retention of TrkA in the Golgi apparatus and inhibit its surface sorting. Taken together, our data provide evidence that Mint2 is a novel TrkA-regulating protein that affects NGF-induced neurite outgrowth, possibly through a mechanism involving retention of TrkA in the Golgi apparatus.

Protein isolation from ear wax made easy

Cerumen is a waxy substance with a mixture of different lipids and and not yet identified proteins. Analysing ear wax can be quite laborious because of the different and sometimes interfering components. Therefore, time-consuming techniques such as chromatography or spectrometry were used to gain informations about the components of ear wax. Conclusions were drawn from immunohistochemical detections of special proteins within the skin or the glands of the external ear canal about the existence of these proteins within the ear wax. But directly analysing the proteins within the ear wax was difficult. We, therefore, worked out a method to isolate proteins from ear wax. Ear wax was collected from 16 adults with no infections of the external ear canal. The protein isolation was conducted using the Qproteome Mammalian Protein Prep Kit by Qiagen in two different kind of ways (cell and lysat fraction). Afterwards, we performed a quantification of the total protein concentration using the BCA method. There was a statistical significant difference in the total protein concentration between the two different (cell and lysat fraction) described ways. Furthermore, it is a fast and easy method to extract proteins from ear wax. The benefit of the described method and the field of application will be discussed.

Mutant Huntingtin N-terminal Fragments of Specific Size Mediate Aggregation and Toxicity in Neuronal Cells

Huntingtin proteolysis is implicated in Huntington disease pathogenesis, yet, the nature of huntingtin toxic fragments remains unclear. Huntingtin undergoes proteolysis by calpains and caspases within an N-terminal region between amino acids 460 and 600. We have focused on proteolytic steps producing shorter N-terminal fragments, which we term cp-1 and cp-2 (distinct from previously described cp-A/cp-B). We used HEK293 cells to express the first 511 residues of huntingtin and further define the cp-1 and cp-2 cleavage sites. Based on epitope mapping with huntingtin-specific antibodies, we found that cp-1 cleavage occurs between residues 81 and 129 of huntingtin. Affinity and size exclusion chromatography were used to further purify huntingtin cleavage products and enrich for the cp-1/cp-2 fragments. Using mass spectrometry, we found that the cp-2 fragment is generated by cleavage of huntingtin at position Arg(167). This site was confirmed by deletion analysis and specific detection with a custom-generated cp-2 site neo-epitope antibody. Furthermore, alterations of this cleavage site resulted in a decrease in toxicity and an increase in aggregation of huntingtin in neuronal cells. These data suggest that cleavage of huntingtin at residue Arg(167) may mediate mutant huntingtin toxicity in Huntington disease.

Tumor-targeted PE38KDEL delivery via PEGylated anti-HER2 immunoliposomes

We previously reported the development of PE38KDEL-loaded anti-HER2 poly(lactic-co-glycolic acid) (PLGA) nanoparticles that bind and internalize in HER2-overexpressing breast cancer cells, enabling potent anti-tumor activity. To overcome the problems associated with the short half-lives of this drug delivery system, we have constructed PE38KDEL-loaded anti-HER2 PEGylated liposomes (PE-HER-liposomes). PE-HER-liposomes were constructed with Fab′ of recombinant humanized anti-HER2 monoclonal antibody (anti-HER2 Fab′) covalently linked to PEGylated liposomes containing PE38KDEL (PE-liposomes). We attached anti-HER2 Fab′ to the terminus of PEG (polyethylene glycol) on PEGylated liposomes. Incorporation of pyridylthiopropionoylamino-PEG-distearoylphosphatidylethanolamine (PDP-PEG-DSPE) into PEGylated liposomes followed by mild thiolysis of the PDP groups resulted in the formation of reactive thiol groups at the periphery of the liposomes. Efficient attachment of maleimide-derivatized anti-HER2 Fab′ took place under mild conditions. The characterization of PE-HER-liposomes, such as particle size, was evaluated by dynamic light-scattering detector. The Micro BCA method was used to determine the encapsulation efficiency of PE38KDEL and the quantity of conjugated Fab′. Flow cytometry and confocal microscopy showed that PE-HER-liposomes possessed receptor-specific binding and internalization for HER2-overexpressing SK-BR3 cells. Remarkably, PE-HER-liposomes were more cytotoxic than non-targeted PE-liposomes in HER2-overexpressing breast cancer cells. In conclusion, PE-HER-liposomes could serve as a promising therapeutic candidate for the treatment of HER2-overexpressing breast cancers.

Revisiting Qualms about Bootstrap Confidence Intervals

SYNOPTIC ABSTRACTIn a rather important paper Schenker (1985) used a particular chi-square distribution for a sample variance to show that the percentile method bootstrap and even the BC bootstrap break down for very practical sample sizes. This caused Efron (1987) to devise the BCa method in a very well-known paper. This paper revisits the issues surrounding bootstrap confidence intervals by looking at a particularly difficult problem, estimating variances in a nonparametric setting. We show by simulation methods that for certain heavy-tailed and skewed sampling distributions for the observed data, the convergence of even the second order accurate bootstrap methods BCa. and ABC, must be slow because even at a sample size of nSize=200 the confidence level is not close to the advertised and correct asymptotic level (e.g. for Student's t with 5 degrees of freedom the methods compared are between 4 and 5% below their nominal levels). At nSize =25, all of the methods provide true confidence levels that are at least 5% below their nominal confidence levels. We illustrate this by using confidence levels of 50%, 75% and 90% among others. To investigate more deeply the convergence properties, we took nSize=1000 or more (i.e. number of observations in the original data set). To adequately show the pattern of convergence, the standard deviation of the Monte Carlo approximation of the proportion needs to be around 0.005. But to achieve this requires close to nRepI =10,000 iterations (i.e. Monte Carlo replications of the bootstrap estimates) of the simulated results! For the high order bootstraps at nSize=1000 and nRepl =10,000 computations become prohibitively intensive even on a fast modem computer! It is interesting that for these simulations the first order percentile method bootstrap did nearly as well as. and sometimes better than, the higher order bootstraps.

High-Performance Immunolatex Possessing a Mixed-PEG/Antibody Coimmobilized Surface: Highly Sensitive Ferritin Immunodiagnostics

To create a high-performance immunoassay system based on a nanosphere/antibody complex, pentaethylenehexamine-ended poly(ethylene glycol), N6-PEG comprising N6-PEG-5k (M(n) = 6000 g/mol) and N6-PEG-2k (M(n) = 2000 g/mol) was employed as a novel blocking agent to modify the surface of nanospheres. Both the antibody (antiferritin) and the N6-PEG were covalently bonded onto the nanospheres by the linkage of their amino groups with the activated carboxyl groups of those particles. The quantification of antiferritin and tethered N6-PEG polymer was carried out using the copper reduction/bicinchoninic acid reaction (the Micro BCA method). Dynamic-light-scattering (DLS) and electrophoretic mobility (mu(e)) measurements were performed to characterize the nanosphere/antiferritin/N6-PEG complex, which was prepared under various conditions. Simultaneously, the immune response of the complex obtained in this manner was measured by the turbidimetric monitoring method in phosphate buffer (10 mM, pH = 7.4). On the basis of all the results, the optimum conditions for preparing an acceptable nanosphere/antiferritin/N6-PEG complex were determined. Interestingly, compared to the blocking treatment with bovine serum albumin (BSA), which is a well-known blocking agent, surface modification with N6-PEG, especially that using a mixture of N6-PEG-5k and N6-PEG-2k, improved the performance (increased immune response yield and decreased detection limit) of the nanosphere/antiferritin complex to a remarkable degree in both phosphate buffer and 100% fetal bovine serum (FBS), thus significantly demonstrating the potential of the nanosphere/antibody/mixed-PEG complex as central to a high-performance immunoassay system.

Study on Preparation and Properties of Nitric Oxide Releasing Polyurethane

In this study, a novel nitric oxide (NO) endogenously releasing polyurethane (PU) material, i.e. PU-g-L-cysteine(PU-Cys), was synthesized by grafting L-cysteine on the film surface. Fluorescence analysis and a BCA method were used to characterize qualitatively and quantitatively the grafted L-cysteine. The results showed that L-cysteine was successfully immobilized on the PU film surface and the density of L-cysteine grafted was calculated to be 15.3nmol/cm2. The NO releasing behavior study of PU-Cys film demonstrated that endogenous NO could be continuously released from PU-Cys film and the accumulated releasing amount within 3h added up to 1.06nmol/cm2. The releasing rate of NO was well matchable to the self-releasing rate of resting platelets in human body. The results of dynamic blood clotting test and hemolysis ratio measurement showed that PU-Cys film had a better haemocompatibility than PU film, which might be the result of endogenously released NO inhibiting platelet adhesion and thrombus formation. Therefore, PU-Cys with the property of endogenous NO releasing is expected to be an ideal blood compatibility material in clinical application.