Turbidimetry Assay Research Articles

Multimodal assessment of non‐specific hemostatic agents for apixaban reversal

Non-specific hemostatic agents, namely activated prothrombin complex concentrate (aPCC), PCC and recombinant activated factor (F) VII (rFVIIa), can be used, off-label, to reverse the effects of FXa inhibitors in the rare cases of severe hemorrhages, as no approved specific antidote is available. We have evaluated the ability of aPCC, PCC and rFVIIa to reverse apixaban. Healthy volunteer whole blood was spiked with therapeutic or supra-therapeutic apixaban concentrations and two doses of aPCC, PCC or rFVIIa. Tests performed included a turbidimetry assay for fibrin polymerization kinetics analysis, scanning electron microscopy for fibrin network structure observation, thrombin generation assay (TGA), thromboelastometry, prothrombin time and activated partial thromboplastin time. aPCC generated a dense clot constituting thin and branched fibers similar to those of a control without apixaban, increased fibrin polymerization velocity and improved quantitative (endogenous thrombin potential and peak height) as well as latency (clotting and lag times) parameters. Adding PCC also improved the fibrin and increased quantitative parameters, but fibrin polymerization kinetics and latency parameters were not corrected. Finally, rFVIIa improved latency parameters but failed to restore the fibrin network structure, fibrin polymerization velocity and quantitative parameters. aPCC was more effective than PCC or rFVIIa in reversing in vitro the effects of apixaban. aPCC rapidly triggered the development of an apparently normal fibrin network and corrected latency and quantitative parameters, whereas PCC or rFVIIa had only a partial effect.

A Detailed Study on Understanding Glycopolymer Library and Con A Interactions.

Synthetic glycopolymers are important natural oligosaccharides mimics for many biological applications. To develop glycopolymeric drugs and therapeutic agents, factors that control the receptor-ligand interaction need to be investigated. A library of well-defined glycopolymers has been prepared by the combination of copper mediated living radical polymerization and CuAAC click reaction via post-functionalization of alkyne-containing precursor polymers with different sugar azides. Employing Concanavalin A as the model receptor, we explored the influence of the nature and densities of different sugars residues (mannose, galactose, and glucose) on the stoichiometry of the cluster, the rate of the cluster formation, the inhibitory potency of the glycopolymers, and the stability of the turbidity through quantitative precipitation assays, turbidimetry assays, inhibitory potency assays, and reversal aggregation assays. The diversities of binding properties contributed by different clustering parameters will make it possible to define the structures of the multivalent ligands and densities of binding epitopes tailor-made for specific functions in the lectin-ligand interaction. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 2588–2597

Effect of safflower yellow on platelet activating factor mediated platelet activation in patients with coronary heart disease

The platelet aggregation and 5-HT release by washed platelet from coronary heart disease patients following platelet activating factor (PAF) treatment were detected by turbidimetry and O-phthalaldehyde assay. The free calcium concentration in the platelets was measured with the fura-2/AM probe fluorescent technique. Results showed safflower yellow could inhibit the PAF induced washed platelet aggregation and 5-HT release, which were in a safflor-yellow-dose dependent manner. When the PAF was 2.0×10 -9 mol/L, the inhibition rate of platelet aggregation was 26.2%, 41.3%, 58.1%, 81.2%, and the inhibition rate of 5-HT release was 3.7%, 11.9%, 29.9% and 54.4% after treatment with safflower yellow at 0.21, 0.42, 0.85 and 1.69 g/L, respectively. The study concludes safflower yellow can inhibit the PAF induced platelet aggregation, 5-HT release by platelets and elevation of free calcium in platelets.

The Research on the Relationship Between Serum High-Sensitivity C-Reactive Protein and Interleukin-6 in Acute Coronary Syndrome Patients

Objective To investigate the relationship among the inflammatory markers of high-sensitivity Creactive protein(hs-CRP), interleukin-6 (IL-6) and the process of the acute coronary syndrome (ACS).Methods Immunity turbidimetry assay was used to test the serum hs-CRP,and enzyme linked immunosorbent assay (ELISA) was used to test the serum IL-6 in 60 ACS cases[33 cases with acute myocardial infarction(AMI) ,27 cases with unstable angina pectoris(UAP)],45 cases with stable angina pectoris (SAP)and 45 healthy persons. The differences of the serum hs-CRP and the serum IL-6 were compared among the three groups and the correlation between serum hs-CRP and the serum IL-6 was also analyzed in the ACSs. Results The concentrations of the serum hs-CRP and the serum IL-6 in ACSs were both significantly higher than those of SAPs and healthy persons (all P＜0.01).The concentrations of the serum hs-CRP and the serum IL-6 in AMIs were significantly higher than those of UAPs (all P＜0.05). And there was a direct correlation between the serum hs-CRP and the serum IL-6 in ACSs (r=0.836).Conclusion The levels of the serum hs-CRP and the serum IL-6 could be used to judge the stability of the plaques,and there was some direct correlation between them in the ACS process, and they should he valuable to predict the prognosis of ACSs. Key words: Acute coronary syndrome; High-sensitivity C-reactive protein; Interleukin-6; Correlation

C-Reactive Protein and the Risk of Cancer: A Mendelian Randomization Study

Elevated plasma levels of C-reactive protein (CRP), a marker of inflammation, are associated with an increased risk of cancer, but it is unclear whether this association is causal. We examined whether four common single-nucleotide polymorphisms (SNPs) in the CRP gene that are associated with altered plasma CRP levels are causally associated with an increased risk of cancer. The study population included participants in a prospective study (n = 10 215) and a cross-sectional study (n = 36 403) of the adult general population in Denmark, all of whom were genotyped for the CRP SNPs. The association between plasma CRP levels measured by a high-sensitivity turbidimetry assay and the risk of cancer was examined for 8224 participants in the prospective study. The hazard ratio of cancer for a doubling of the plasma CRP level was 1.09 (95% confidence interval [CI] = 1.03 to 1.14). The nine most common genotype combinations of the four CRP SNPs were associated with up to a 72% increase (95% CI = 58% to 87%) in CRP levels but not with an increased risk of cancer. The estimated causal odds ratio for cancer associated with a genetically induced doubling in CRP level was 0.94 (95% CI = 0.81 to 1.08). This finding suggests that elevated CRP levels do not cause cancer.

Effects of Allium hirtifolium (Iranian shallot) and its allicin on microtubule and cancer cell lines

Allium hirtifolim Boiss. (Iranian Shallot) belongs to Allium genus (Alliaceae family). Microtubule proteins (MTs) are crucial in maintenance of cell shape as well as cell division and mitosis. The present study aims at defining the anti-microtubule activities of A. hirtifolium and its allicin and examining its effects on nerve cell microtubules. MTs were prepared from sheep brain through two cycles of polymerization and depolymerization. The cell growth inhibition was measured by 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) after treatment with A. hirtifolium and its allicin on HeLa and MCF-7 and L-929 cell lines. A. hirtifolium displayed growth inhibitory activity against HeLa and MCF-7 cells with IC (50) value of 20 and 24 mg/L, respectively, for 72 h and obviously showed cell growth inhibition on these cell lines at non-toxic concentration (lower than 1 g/L). Inhibition of MTs polymerization induced by A. hirtifolium and its ability to bind to tubulin as a ligand was tested through turbidimetry assay then investigated by Transmission Electron Microscopy (TEM). The concentration of A. hirtifolium necessary to inhibit the assembly of MTs by 50% was 1.2 g/L, while an inhibition higher than 80% was observed in the presence of 4 g/L of A. hirtifolium. This plant decreased MTs polymerization; therefore we suggest A. hirtifolium can be an effective ligand for cancer therapy.

Protective Effects of l- and d-Carnosine on α-Crystallin Amyloid Fibril Formation: Implications for Cataract Disease

Mildly denaturing conditions induce bovine alpha-crystallin, the major structural lens protein, to self-assemble into fibrillar structures in vitro. The natural dipeptide l-carnosine has been shown to have potential protective and therapeutic significance in many diseases. Carnosine derivatives have been proposed as potent agents for ophthalmic therapies of senile cataracts and diabetic ocular complications. Here we report the inhibitory effect induced by the peptide (l- and d-enantiomeric form) on alpha-crystallin fibrillation and the almost complete restoration of the chaperone activity lost after denaturant and/or heat stress. Scanning force microscopy (SFM), thioflavin T, and a turbidimetry assay have been used to determine the morphology of alpha-crystallin aggregates in the presence and absence of carnosine. DSC and a near-UV CD assay evidenced that the structural precursors of amyloid fibrils are polypeptide chain segments that lack stable structural elements. Moreover, we have found a disassembling effect of carnosine on alpha-crystallin amyloid fibrils. Finally, we show the ability of carnosine to restore most of the lens transparency in organ-cultured rat lenses exposed to similar denaturing conditions that were used for the in vitro experiments.

Synthesis of pegylated immunonanoparticles.

This work describes the synthesis of pegylated immunonanoparticles by conjugation of an anti-transferrin receptor monoclonal antibody (MAb) to maleimide-grafted pegylated nanoparticles prepared from poly(lactic acid) (PLA) and a bi-functional polyethyleneglycol (PEG). Maleimide-PEG3500-PLA40000 and methoxyPEG2600-PLA40000 copolymers were synthesized by ring opening polymerization of L-lactide using stannous octoate as catalyst. Pegylated nanoparticles were prepared from these copolymers by a multiple emulsion/solvent evaporation method and thiolated OX26 MAb was conjugated through the maleimide function located at the distal end of the PEG spacer. The pegylated immunonanoparticles were characterized by quasi-elastic light scattering, gel permeation chromatography, turbidimetry assays, and transmission electron microscopy. NMR spectroscopy confirmed the synthesis of both copolymers and the preservation of the maleimide function. The pegylated immunonanoparticles had an average diameter of 121 +/- 5 nm and appeared spherical by transmission electron microscopy. The number of OX26 MAb molecules conjugated per individual pegylated nanoparticle was 67 +/- 4. The MAb conjugated to the surface of the pegylated immunonanoparticle was visualized directly by electron microscopy using a conjugate of 10 nm gold and an anti-mouse immunoglobulin secondary antibody. Pegylated immunonanoparticles can be synthesized with bifunctional PEG derivatives that bridge the nanoparticle and the targeting MAb. This novel formulation may enable the targeted delivery of small molecules, protein drugs, and gene medicines.

EFFECTS OF TAMOXIFEN, MELATONIN, COENZYME Q10, ANDl-CARNITINE SUPPLEMENTATION ON BACTERIAL GROWTH IN THE PRESENCE OF MYCOTOXINS

The involvement of toxic oxygen intermediates in the bacteriostatic effects of mycotoxins (T-2 toxin, deoxynivalenol, ochratoxin A, aflatoxin B1, and fumonisin B1) was investigated by producing bacterial growth curves using turbidimetry assays in the presence and absence of oxygen radical-scavenging substances. The strains used in this study includedEscherichia coli(FT 101),Streptococcus agalactiae(FT 311,FT 313,FT 315),Staphylococcus aureus(FT 192),Yersinia enterocolitica(FT 430),Salmonella infantis(FT 431),Erysipelothrix rhusiopathiae(FT 432),Lactobacillus plantarum(FT234) andLactobacillus casei(FT 232). Tamoxifen, melatonin,l-carnitine and coenzyme Q10were used as radical scavengers against oxygen toxicity to the strains studied. Tamoxifen was the most effective in inhibiting bacterial growth when used at a high concentration, whereas melatonin andl-carnitine were less effective. A combination ofl-carnitine and coenzyme Q10provided better protection against oxygen toxicity caused by the mycotoxins growth than they did individually. It was concluded that oxygen radicals are involved in the killing of bacteria and that there is endogenous formation of toxic oxygen products by mycotoxins. The objective of this study was to determine whether the antioxidants were able to counteract the toxic effects of the mycotoxins. The data obtained indicate that bacterial growth can be inhibited especially by T-2 toxin, aflatoxin B1and ochratoxin A and that this effect can be partially counteracted by antioxidants such as coenzyme Q10plusl-carnitine.

Tubulointerstitial nephritis antigen interacts with laminin and type IV collagen and promotes cell adhesion.

Tubulointerstitial nephritis (TIN) antigen has been recently identified as a novel basement membrane macromolecule. It consists of a single chain of 58 kDa and exhibits a restricted distribution. The interaction between TIN antigen and laminin or type IV collagen has been studied using solid-phase binding assays and found to be for both macromolecules specific, saturable, and with an affinity in the low micromolar range. In similar assays, TIN antigen did not interact with heparin. In turbidimetry assays, it was found that the presence of TIN antigen did not affect the polymerization of type IV collagen but had a concentration-dependent inhibitory effect on laminin polymerization and on preformed laminin polymers. TIN antigen was able to promote adhesion of epithelial cells derived from kidney tubules and of endothelial cells derived from aorta. The data suggest that TIN antigen may be a macromolecule of importance both for basement membrane ultrastructure and cellular adhesion.