In Vitro and In Vivo Mouse Models of the Type 1 Von Willebrand Disease Mutations Y1584C and R1205H.

Abstract 26 Introduction: Type 1 von Willebrand disease (VWD) is caused by mutations that result in moderate decreases in VWF (5-50% of normal levels) and a mild bleeding phenotype. The VWF missense mutation Y1584C is associated with mildly decreased VWF:Ag levels, increased ADAMTS13 cleavage, as well as a possible increase in clearance. The Vicenza mutation, R1205H, exhibits a more severe phenotype (VWF:Ag ∼10%), as well as accelerated clearance. However, extensive controlled in vitro and in vivo investigation of these mutations has yet to be described. In this study, we examine both Y1584C and R1205H in comparison to wild type VWF using in vitro and in vivo strategies employing both human and mouse VWF. Methods: Recombinant murine and human VWF and ADAMTS13 were produced via transient transfection in HEK293T cells in serum free OPTIMEM for 72 hours. Full length ADAMTS13 digests were performed in a Tris-Urea system and analyzed via multimer pattern. VWF115 digests were performed in an ELISA based assay. Hydrodynamic injections were performed using 100 μg wild type (WT) or mutant ET-mVWF plasmid DNA in Ringer's solution in 7-9 week old C57Bl6 VWF knockout mice. Mice were sampled at days 2, 5, 8, and then weekly. Mouse plasma was analyzed for CBCs, VWF:Ag, and VWF multimer structure. Results: In the HEK293T transient transfection system, secreted mutant protein was similar to that of wild type recombinant protein, with high molecular weight material present. ADAMTS13 digestion of full length recombinant Y1584C versus wild type showed no statistical difference: 50% cleavage for hVWF WT 1.78U hADAMTS13, hVWF Y1584C 1.67 U, P=0.58; mVWF WT 0.32 U mADAMTS13; Y1584C 0.42 U, P=0.11. In contrast, the Y1584C substitution in the hVWF115 construct required 40% less hADAMTS13 to effect equivalent cleavage (WT 0.087±0.014, 4; Y1584C 0.052±0.005, 8, mean U/ml ADAMTS13±SEM, N, P=0.013). Mouse ADAMTS13 cleavage of mVWF115 was also increased 20% for Y1584C (WT: 0.20±0.06, 4; Y1584C: 0.16±0.01, 4, P=0.014). Hydrodynamic injection caused no adverse events in any animals. CBC values were not statistically significantly different between wild type and mutants. Initial high VWF:Ag values were similar for wild type VWF (25.6±2.9, 15, mean U/ml±SEM, n) and Y1584C (27.2±5.0, 11), but R1205H levels were 36% lower (16.3±2.1, 10). At 14 days, WT VWF:Ag was 5.33±1.13, 15, with R1205H (1.73±0.44, 12) and Y1584C (1.84±0.59, 11) VWF:Ag levels being 68% and 65% lower, respectively. R1205H continued to remain approximately 40% of WT values for the next three weeks, while Y1584C continued to decrease, dropping to 15% on day 21 and 8% on day 28, compared to the wild type values at these time points. The R1205H mutation showed no significant difference in multimer structure defined by observed number of bands (days 2 to 42, mean difference -0.49, P>0.05) to wild type VWF. In contrast, Y1584C had a significant decrease in band number (3.38, P<0.001). Conclusions: This study demonstrates that these two type 1 VWD mutations have a strong observable effect in the VWF knockout mouse model. R1205H exhibits a large decrease in VWF:Ag levels at all measured time points, but no alteration in multimer structure. Y1584C, in contrast, shows a loss of high molecular weight material, and an initially high VWF:Ag level that rapidly decays from day 14 onward, suggesting increased ADAMTS13 cleavage and increased clearance. In addition, in vitro ADAMTS13 testing shows that Y1584C responds differently in the two assay systems with only the VWF115 assay showing significant increases in ADAMTS13-mediated cleavage. Disclosures: No relevant conflicts of interest to declare.

Quantification of Muconic Acid-Type Structures in High Molecular Weight Material from Bleach Plant Effluents

Summary Effluents from the chlorine dioxide bleaching of chemical pulps were characterized with respect to high and low molecular weight material, COD, TOC, elemental composition, and carboxylic acid and methyl ester content. A technique was developed for the quantitative estimation of muconic acid-type structures as moities of the high molecular weight material. The method is based on the use of quantitative 13C NMR and the pH-dependent cyclization of muconic acids in aqueous solution. 20–40 % of the carboxylic acids in the high molecular weight material isolated from bleach plant effluents from three softwood kraft mills were found to exhibit muconic acid-type behaviour.

Removal of Natural Organic Matter (NOM) from Drinking Water Supplies by Ozone-Biofiltration

Removal of natural organic matter (NOM) in biofilters can be affected by many factors including NOM characteristics, use of pre-ozonation, water temperature, and biofilter backwashing. Laboratory experiments were performed and a biofilter simulation model was developed for the purpose of evaluating the effects of each of these factors on NOM removal in biofilters. Four sources of NOM were used in this study to represent a broad spectrum of NOM types that may be encountered in water treatment. In batch experiments with raw NOM, the removal of organic carbon by biodegradation was inversely proportional to the UV absorbance (254 nm)-to-TOC ratio and directly proportional to the percentage of low molecular weight material (as determined by ultrafiltration). The extent and rate of total organic carbon (TOC) removal typically increased as ozone dose increased, but the effects were highly dependent on NOM characteristics. NOM with a higher percentage of high molecular weight material experienced the greatest enhancement in biodegradability by ozonation. The performance of laboratory-scale continuous-flow biofilters was not significantly affected by periodic backwashing, because backwashing was unable to remove large amounts of biomass from the filter media. Model simulations confirmed our experimental results and the model was used to further evaluate the effects of temperature and backwashing on biofilter performance.

Poly(2-Butyne-1,4-Diyl)S as Precursors to Novel Substituted Polyacetylenes

Thermally-induced polymerization of 1,4-diphenyl-1,2,3-butatriene affords poly(1,4-diphenyl- 2-butyne-1,4-diyl) as a soluble high molecular weight material. Structure is characterized by 13C and vibrational spectroscopy. When treated with base, this material rearranges to a soluble red poly(acetylene) with a unique substitution pattern.

Copper(I)-catalyzed cross-dehydrocoupling reactions of silanes and amines

In the presence of cuprous chloride, hydrosilanes and primary amines react to give silazanes. Methylphenylsilane reacts with (R)-(+)-α-methylbenzylamine to give the two diastereomeric monosilazanes as the only products. Aniline and benzylamine react with methylphenyl- and diphenylsilanes to give primarily the monosilazane, disilazane, or diazasilane as the main product, depending on the reactant ratios. Phenylsilane gives higher oligomers with aniline, mainly the trisilatriazane and trisiladiazanes, but no high molecular weight material. Keywords: silanes, amines, silazanes, cuprous chloride, dehydrocoupling, catalysis.

An improved method for the rapid isolation of chromosomal DNA from Mycoplasma spp.

A method is described for the rapid isolation of chromosomal deoxyribonucleic acid from species of the genus Mycoplasma. The method involves incubation of washed cells at elevated temperature in the presence of an ionic detergent, chelating agents, and proteinase K prior to the removal of residual protein and ribonucleic acid with ribonuclease and chloroform. It results in a good yield of high molecular weight material that is shown to be free of endogenous nuclease and substantially free of protein or ribonucleic acid contamination without the use of phenol. The isolated DNA is shown to be an excellent substrate for restriction endonuclease digestion and ligation with T4 DNA ligase. Key words: Mycoplasma, DNA isolation, micromethod.