Optimal quality 131I-monoclonal antibodies on high-dose labeling in a large reaction volume and temporarily coating the antibody with IODO-GEN

A novel, facile procedure for efficient coupling of high doses of 131I to monoclonal antibodies (MAbs) was developed with minimal chemical and radiation damage. Methods: To diminish the radiation and chemical burden during labeling, iodination was performed in a large reaction volume and by temporarily coating the MAb with a minimal amount of IODO-GEN. The MAb was coated by injection of IODO-GEN (dissolved in acetonitrile [MeCN]) into the aqueous MAb solution, and the coating was subsequently removed by addition of ascorbic acid. For chemoprotection before, during, and after PD-10 purification of the 131I-MAbs, ascorbic acid and human serum albumin were used. The effects of autoradiolysis in the starting 131I solution were countered by treatment with NaOH and ascorbic acid. For this so-called IODO-GEN-coated MAb method, the sensitive chimeric MAb MOv18 (c-MOv18) and the more robust murine MAbs K928 and E48 were used. The high-dose 131I-labeled MAbs were characterized for radiochemical purity and MAb integrity by thin-layer chromatography, high-performance liquid chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by phosphor imager quantification. The high-dose 131I-labeled MAbs were also characterized for immunoreactivity. The radiopharmacokinetics and biodistribution of 131I-c-MOv18 were analyzed in human tumor-bearing nude mice. For comparison, 131I-c-MOv18 batches were made using the conventional chloramine-T or IODO-GEN-coated vial method. Results: Conventional high-dose labeling of 5 mg c-MOv18 with 4.4 GBq 131I resulted in a labeling yield of 60%, a radiochemical purity of 90%, an immunoreactive fraction of 25% (72% being the maximum in the assay used), and the presence of aggregation and degradation products. Using similar amounts of 131I and MAb in the IODO-GEN-coated MAb method, 85%-89% overall radiochemical yield, at least 99.7% radiochemical purity, and full preservation of MAb integrity and immunoreactivity were achieved. For this labeling, 5 mg MAb were coated with 35 μg IODO-GEN during 3 min in a reaction volume of 6 mL. Also, biodistribution was optimal, and tumor accumulation was superior to that of coinjected 125I-c-MOv18 labeled according to the conventional IODO-GEN-coated vial method. Conclusion: A new, facile, high-dose 131I-labeling method was developed for production of 131I-labeled MAbs with optimal quality for use in clinical radioimmunotherapy

Molecular evidence for putative tumour suppressor genes on chromosome 13q specific to BRCA1 related ovarian and fallopian tube cancer

Background/Aims: Loss of heterozygosity (LOH) on chromosome 13q has been reported to occur frequently in human ovarian cancer, and indications have been found that chromosome 13 may also play a specific role in the inherited form of ovarian cancer. The aim of this study was to define regions on chromosome 13 that may harbour additional tumour suppressor genes involved in the tumorigenesis of BRCA1 related ovarian and fallopian tube cancer. Materials/methods: DNA extracted from paraffin wax blocks of 36 BRCA1 associated ovarian and fallopian tube carcinomas was analysed by LOH polymerase chain reaction using seven highly polymorphic microsatellite markers spanning chromosome 13q. Results: High LOH frequencies were found on loci 13q11, 13q14, 13q21, 13q22–31, 13q32, and 13q32–4, suggesting the presence of putative tumour suppressor genes on the long arm of chromosome 13 that may play a role in the pathogenesis of BRCA1 related ovarian and fallopian tube cancer. LOH patterns appeared to be independent of the type of BRCA1 mutation, stage, and grade. Although in some cases there were indications for loss of larger parts of chromosome 13, in most cases losses were fairly randomly distributed over chromosome 13 with retained parts in between lost parts. Microsatellite instability was found in six cases. Conclusion: Several loci on chromosome 13q show high frequencies of LOH in BRCA1 related ovarian and fallopian tube cancer, and may therefore harbour putative tumour suppressor genes involved in the carcinogenesis of this particular type of hereditary cancer

No ion is an island: Multiple ions influence boron incorporation into CaCO3

Boron isotope ratios – as measured in marine calcium carbonate – are an established tracer of past seawater and calcifying fluid pH, and thus a powerful tool for probing marine calcifier physiology and reconstructing past atmospheric CO 2 levels. For such applications, understanding the inorganic baseline upon which foraminiferal vital effects or coral pH upregulation are superimposed should be an important prerequisite. Yet, investigations into boron isotope fractionation in synthetic CaCO 3 polymorphs have often reported variable and even conflicting results, implying our understanding of the pathways of boron incorporation into calcium carbonate is incomplete. Here we address this topic with experimental data from synthetic calcite and aragonite precipitated across a range of pH in the presence of both Mg and Ca. We observe coherent patterns in B/Ca and Na/Ca ratios that, we suggest, point to paired substitution of Na and B into the carbonate lattice to achieve local charge balance. In addition, we confirm the results of previous studies that the boron isotope composition of inorganic aragonite precipitates closely reflects that of aqueous borate ion, but that inorganic calcites display a higher degree of scatter, and diverge from the boron isotope composition of aqueous borate ion at low pH. With reference to the simultaneous incorporation of other trace and minor elements, we put forward possible explanations for the observed variability in the concentration and isotopic composition of boron in synthetic CaCO 3. In particular, we highlight the potential importance of interface electrostatics in driving variability in our own and published synthetic carbonate datasets. Importantly for palaeo-reconstruction, however, these electrostatic effects are unlikely to play as important a role during natural precipitation of biogenic carbonates. </p

Proteomic Screen Identifies IGFBP7 as a Novel Component of Endothelial Cell-Specific Weibel-Palade Bodies

Vascular endothelial cells contain unique storage organelles, designated Weibel-Palade bodies (WPBs), that deliver inflammatory and hemostatic mediators to the vascular lumen in response to agonists like thrombin and vasopressin. The main component of WPBs is von Willebrand factor (VWF), a multimeric glycoprotein crucial for platelet plug formation. In addition to VWF, several other components are known to be stored in WPBs, like osteoprotegerin, monocyte chemoattractant protein-1 and angiopoetin-2 (Ang-2). Here, we used an unbiased proteomics approach to identify additional residents of WPBs. Mass spectrometry analysis of purified WPBs revealed the presence of several known components such as VWF, Ang-2, and P-selectin. Thirty-five novel candidate WPB residents were identified that included insulin-like growth factor binding protein-7 (IGFBP7), which has been proposed to regulate angiogenesis. Immunocytochemistry revealed that IGFBP7 is a bona fide WPB component. Cotransfection studies showed that IGFBP7 trafficked to pseudo-WPB in HEK293 cells. Using a series of deletion variants of VWF, we showed that targeting of IGFBP7 to pseudo-WPBs was dependent on the carboxy-terminal D4-C1-C2-C3-CK domains of VWF. IGFBP7 remained attached to ultralarge VWF strings released upon exocytosis of WPBs under flow. The presence of IGFBP7 in WPBs highlights the role of this subcellular compartment in regulation of angiogenesis