Enhancing immunoassay detection of antigens with multimeric protein Gs

This paper describes a method for the effective and self-oriented immobilization of antibodies on magnetic silica-nanoparticles using a multimeric protein G. Cysteine-tagged recombinant dimers and trimers of protein G were produced in Escherichia coli BL21 by repeated linking of protein G monomers with a flexible (GGGGS)(3) linker. Amino-functionalized silica-coated magnetic nanoparticles (SiO(2)-MNPs, Fe(3)O(4)@SiO(2)) were prepared and coupled to the protein G multimers, giving the final magnetic immunosensor. The optimal conditions for the reaction between the protein Gs and the SiO(2)-MNPs was a time of 60 min and a concentration of 100 μg/mL, resulting in coupling efficiencies of 77%, 67% and 55% for the monomeric, dimeric and trimeric protein Gs, respectively. Subsequently, anti-hepatitis B surface antigen (HBsAg) was immobilized onto protein G-coupled SiO(2)-MNPs. The quantitative efficiency of antibody immobilization found the trimeric protein G to be the best, followed by the dimeric and monomeric proteins, which differs from the coupling efficiencies. Using all three protein constructs in an HBsAg fluoroimmunoassay, the lowest detectable concentrations were 500, 250 and 50 ng/mL for the monomeric, dimeric and trimeric protein G-coupled SiO(2)-MNPs, respectively. Therefore, multimeric protein Gs, particularly the trimeric form, can be employed to improve antibody immobilization and, ultimately, enhance the sensitivity of immunoassays. In addition, the multimeric protein Gs devised in this study can be utilized in other immunosensors to bind the antibodies at a high efficiency and in the proper orientation.     

Molecular identification of AFLP fragments associated with elytra color variation of the Asian ladybird beetle,Harmonia axyridis (Coleoptera: Coccinellidae)

The Asian ladybird beetle, Harmonia axyridis shows polymorphism in elytra color patterns. However, it is uncertain whether these color patterns are regulated by genetic factors. This investigation used amplified fragment length polymorphism (AFLP) analysis to determine any genetic causes of the variability of color patterns. Using four individuals of each group, AFLP analysis produced 37 polymorphic bands. Among several polymorphic bands, six AFLP markers were associated with elytra color patterns after further analysis using six additional individuals of each group. These polymorphic sites were sequenced but did not match DNA sequence data deposited in GenBank. Based on the color-associated AFLP markers, SCAR primers were designed for PCR amplification of genomic DNA. These primers (SCAR 12 and SCAR 44) were used to analyze color-associated loci and/or alleles of H. axyridis DNA. SCAR 12 primers designed from a Spectabilis type-specific fragment (AFLP 12) amplified a specific band of 530 bp in four Spectabilis individuals, but not in the insects with other color patterns.     

Angiopoietin-1 protects heart against ischemia/reperfusion injury through VE-cadherin dephosphorylation and myocardiac integrin-β1/ERK/caspase-9 phosphorylation cascade

Early reperfusion after myocardial ischemia that is essential for tissue salvage also causes myocardial and vascular injury. Cardioprotection during reperfusion therapy is an essential aspect of treating myocardial infarction. Angiopoietin-1 is an endothelial-specific angiogenic factor. The potential effects of angiopoietin-1 on cardiomyocytes and vascular cells undergoing reperfusion have not been investigated. We propose a protective mechanism whereby angiopoietin-1 increases the integrity of the endothelial lining and exerts a direct survival effect on cardiomyocytes under myocardial ischemia followed by reperfusion. First, we found that angiopoietin-1 prevents vascular leakage through regulating vascular endothelial (VE)-cadherin phosphorylation. The membrane expression of VE-cadherin was markedly decreased on hypoxia/reoxygenation but was restored by angiopoietin-1 treatment. Interestingly, these effects were mediated by the facilitated binding between SH2 domain-containing tyrosine phosphatase (SHP2) or receptor protein tyrosine phosphatase μ (PTPμ) and VE-cadherin, leading to dephosphorylation of VE-cadherin. siRNA against SHP2 or PTPμ abolished the effect of angiopoietin-1 on VE-cadherin dephosphorylation and thereby decreased levels of membrane-localized VE-cadherin. Second, we found that angiopoietin-1 prevented cardiomyocyte death, although cardiomyocytes lack the angiopoietin-1 receptor Tie2. Angiopoietin-1 increased cardiomyocyte survival through integrin-β1-mediated extracellular signal-regulated kinase (ERK) phosphorylation, which inhibited caspase-9 through phosphorylation at Thr12? and subsequently reduced active caspase-3. Neutralizing antibody against integrin-β1 blocked these protective effects. In a mouse myocardial ischemia/reperfusion model, angiopoietin-1 enhanced cardiac function and reduction in left ventricular-end systolic dimension (LV-ESD) and left ventricular-end diastolic dimension (LV-EDD) with an increase in ejection fraction (EF) and fractional shortening (FS). Our findings suggest the novel cardioprotective mechanisms of angiopoietin-1 that are achieved by reducing both vascular leakage and cardiomyocyte death after ischemia/reperfusion injury.     

Enzyme precipitate coatings of lipase on polymer nanofibers

Lipase (LP) was immobilized on electrospun and ethanol-dispersed polystyrene–poly(styrene-co-maleic anhydride) (PS–PSMA) nanofibers (EtOH-NF) in the form of enzyme precipitate coatings (EPCs). LP precipitate coatings (EPCs-LP) were prepared in a three-step process, consisting of covalent attachment, LP precipitation, and crosslinking of precipitated LPs onto the covalently attached LPs via glutaraldehyde treatment. The LP precipitation was performed by adding various concentrations of ammonium sulfate (20–50%, w/v). EPCs-LP improved the LP activity and stability when compared to covalently attached LPs (CA-LP) and the enzyme coatings of LPs (EC-LP) without the LP precipitation. For example, the use of 40% (w/v) ammonium sulfate resulted in EPC40-LP with the highest activity, which was 4.0 and 3.6 times higher than those of CA-LP and EC-LP, respectively. After 165-day incubation under rigorous shaking at 200 rpm, the residual activities of EPC50-LP were 0.5 μM/min mg of EtOH-NF, representing 113 and 75 times higher than those of CA-LP and EC-LP, respectively. When LP was partially purified via a simple ammonium sulfate precipitation and dialysis, both activities and stabilities of EC-LP and EPC-LP could be marginally improved. It is anticipated that the improved LP activity and stability in the form of EPCs would allow for their potential applications in various bioconversion processes such as biodiesel production and ibuprofen resolution.     

Selective tumor targeting by enhanced permeability and retention effect. Synthesis and antitumor activity of polyphosphazene-platinum (II) conjugates

Nanosized polyphosphazene-platinum (II) conjugates with a wide range of molecular weight from 24,000 to 115,000 were synthesized to study their tumor selectivity by enhanced permeability and retention (EPR) effect and their antitumor activity. It has been found from biodistribution study that the present polyphosphazene-Pt(II) conjugates exhibit high tumor selectivity by EPR effect with the tumor to tissue ratio (TTR) from 3.6 to 13 depending on the molecular size. These polymer conjugates have shown excellent in vivo antitumor activity against both murine and human cancer cell lines. In particular, xenograft trials of the conjugates have shown outstanding tumor inhibition effect on the stomach cancer cell line, YCC-3, which is one of the least responsive to the anticancer agents currently in clinical use, although the reason is not clearly explainable yet. The high in vivo activity seems to be attributed to the controlled-release of the antitumor active platinum (II) moiety, [GlyGluPt(dach] (dach=trans-(+/-)-1,2-diaminocyclohexane) from the phosphazene backbone by degradation in aqueous solution.     

Synthesis, characterization, and pharmacokinetic studies of PEGylated glucagon-like peptide-1

Glucagon-like peptide-1-(7-36) (GLP-1) is a hormone derived from the proglucagon molecule, which is considered a highly desirable antidiabetic agent mainly due to its unique glucose-dependent stimulation of insulin secretion profiles. However, the development of a GLP-1-based pharmaceutical agent has a severe limitation due to its very short half-life in plasma, being primarily degraded by dipeptidyl peptidase IV (DPP-IV) enzyme. To overcome this limitation, in this article we propose a novel and potent DPP-IV-resistant form of a poly(ethylene glycol)-conjugated GLP-1 preparation and its pharmacokinetic evaluation in rats. Two series of mono-PEGylated GLP-1, (i) N-terminally modified PEG(2k)-N(ter)-GLP-1 and (ii) isomers of Lys(26), Lys(34) modified PEG(2k)-Lys-GLP-1, were prepared by using mPEG-aldehyde and mPEG-succinimidyl propionate, respectively. To determine the optimized condition for PEGylation, the reactions were monitored at different pH buffer and time intervals by RP-HPLC and MALDI-TOF-MS. The in vitro insulinotropic effect of PEG(2k)-Lys-GLP-1 showed comparable biological activity with native GLP-1 (P = 0.11) in stimulating insulin secretion in isolated rat pancreatic islet and was significantly more potent than the PEG(2k)-N(ter)-GLP-1 (P < 0.05) that showed a marked reduced potency. Furthermore, PEG(2k)-Lys-GLP-1 was clearly resistant to purified DPP-IV in buffer with 50-fold increased half-life compared to unmodified GLP-1. When PEG(2k)-Lys-GLP-1 was administered intravenously and subcutaneously into rats, PEGylation improved the half-life, which resulted in substantial improvement of the mean plasma residence time as a 16-fold increase for iv and a 3.2-fold increase for sc. These preliminary results suggest a site specifically mono-PEGylated GLP-1 greatly improved the pharmacological profiles; thus, we anticipated that it could serve as potential candidate as an antidiabetic agent for the treatment of non-insulin-dependent diabetes patients.     

Mammalian PIG-X and yeast Pbn1p are the essential components of glycosylphosphatidylinositol-mannosyltransferase I

Within the endoplasmic reticulum (ER), mannoses and glucoses, donated from dolichol-phosphate-mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using dolichol-phosphate-monosaccharide.