Spontaneous Irs1 passenger mutation linked to a gene-targeted SerpinB2 allele

In characterizing mice with targeted disruption of the SerpinB2 gene, we observed animals that were small at birth with delayed growth and decreased life expectancy. Although this phenotype cosegregated with homozygosity for the inactive SerpinB2 allele, analysis of homozygous SerpinB2-deficient mice derived from two additional independent embryonic stem (ES) cell clones exhibited no growth abnormalities. Examination of additional progeny from the original SerpinB2-deficient line revealed recombination between the small phenotype (smla) and the SerpinB2 locus. The locus responsible for smla was mapped to a 2.78-Mb interval approximately 30 Mb proximal to SerpinB2, bounded by markers D1Mit382 and D1Mit216. Sequencing of Irs1 identified a nonsense mutation at serine 57 (S57X), resulting in complete loss of IRS1 protein expression. Analysis of ES cell DNA suggests that the S57X Irs1 mutation arose spontaneously in an ES cell subclone during cell culture. Although the smla phenotype is similar to previously reported Irs1 alleles, mice exhibited decreased survival, in contrast to the enhanced longevity reported for IRS1 deficiency generated by gene targeting. This discrepancy could result from differences in strain background, unintended indirect effects of the gene targeting, or the minimal genetic interference of the S57X mutation compared with the conventionally targeted Irs1-KO allele. Spontaneous mutations arising during ES cell culture may be a frequent but underappreciated occurrence. When linked to a targeted allele, such mutations could lead to incorrect assignment of phenotype and may account for a subset of markedly discordant results from experiments independently targeting the same gene

The endothelial-specific regulatory mutation, Mvwf1, is a common mouse founder allele

Mvwf1 is a cis-regulatory mutation previously identified in the RIIIS/J mouse strain that causes a unique tissue-specific switch in the expression of an N-acetylgalactosaminyltransferase, B4GALNT2, from intestinal epithelium to vascular endothelium. Vascular B4galnt2 expression results in aberrant glycosylation of von Willebrand Factor (VWF) and accelerated VWF clearance from plasma. We now report that 13 inbred mouse strains share the Mvwf1 tissue-specific switch and low VWF phenotype, including five wild-derived strains. Genomic sequencing identified a highly conserved 97-kb Mvwf1 haplotype block shared by these strains that encompasses a 30-kb region of high nucleotide sequence divergence from C57BL6/J flanking B4galnt2 exon 1. The analysis of a series of bacterial artificial chromosome (BAC) transgenes containing B4galnt2 derived from the RIIIS/J or C57BL6/J inbred mouse strains demonstrates that the corresponding sequences are sufficient to confer the vessel (RIIIS/J) or intestine (C57BL6/J)-specific expression patterns. Taken together, our data suggest that the region responsible for the Mvwf1 regulatory switch lies within an approximately 30-kb genomic interval upstream of the B4galnt2 gene. The observation that Mvwf1 is present in multiple wild-derived strains suggests that this locus may be retained in wild mouse populations due to positive selection. Similar selective pressures could contribute to the high prevalence of von Willebrand disease in humans

Contamination of Chalk groundwater by chlorinated solvents : a case study of deep penetration by non-aqueous phase liquids

The transport behaviour of chlorinated solvents, both in the aqueous phase and as a dense non-aqueous phase liquid (DNAPL), in fissured microporous aquifers is reviewed. The presence of DNAPL in aquifers is especially serious as it is likely to be the main subsurface source of contamination and, given the slow rates of dissolution in groundwater, may persist for decades. However, the identification and quantification of DNAPLs in fractured aquifers present many practical problems and are often not achievable. A case study of a Chalk site which had been contaminated by chlorinated solvents demonstrated that the use of a range of techniques, including depth profiling of solvent porewater concentrations in cored boreholes, can provide clear evidence for the presence of DNAPL at depth, although DNAPL was not itself observed. Theoretical considerations and field observations confirmed that DNAPL movement is via fractures rather than through the microporous matrix

Activated Platelets in Carotid Artery Thrombosis in Mice Can Be Selectively Targeted with a Radiolabeled Single-Chain Antibody

BACKGROUND: Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis. METHODOLOGY/PRINCIPAL FINDINGS: LIBS as well as an unspecific control single-chain antibody were labeled with (111)Indium ((111)In) via bifunctional DTPA ( = (111)In-LIBS/(111)In-control). Autoradiography after incubation with (111)In-LIBS on activated platelets in vitro (mean 3866 ± 28 DLU/mm(2), 4010 ± 630 DLU/mm(2) and 4520 ± 293 DLU/mm(2)) produced a significantly higher ligand uptake compared to (111)In-control (2101 ± 76 DLU/mm(2), 1181 ± 96 DLU/mm(2) and 1866 ± 246 DLU/mm(2)) indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of (111)In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630 ± 10650 DLU/mm(2) vs. 17390 ± 7470 DLU/mm(2); P<0.05). These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with (111)In-LIBS resulted in a significant increase of the target-to-background ratio compared to (111)In-control (1.99 ± 0.36 vs. 1.1 ± 0.24; P < 0.01). CONCLUSIONS/SIGNIFICANCE: Nuclear imaging with (111)In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of activated platelets in plaque pathology and atherosclerosis and might be of interest for further developments towards clinical application

Chemiluminescence in activated human neutrophils

Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (HBSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), luminol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUCDCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radish peroxidase (HRP) was added. Both CL and LDCL depend on the generation of Superoxide and on myeloperoxidase (MPO). Treatment of PMNs with azide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, or detapac generated very little CL upon addition of HRP, suggesting that CL is the: result of the interaction among H 2 O 2 , a peroxidase, and trace metals. In a cell-free system practically no CL was generated when H 2 O 2 was mixed with HRP in distilled water (DW). On the other hand significant CL was generated when either HBSS or RPMI media was employed. In both cases CL was markedly depressed either by deferoxamine or by EDTA, suggesting that these media might be contaminated by trace metals, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buffers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn 2+ , Fe 2+ , Cu 2+ , and Co 2+ very markedly enhanced CL induced by mixtures of H 2 O 2 and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salts in HBSS and RPMI interfered with the activity of the added metals. CL and LDCL of activated PMNs was enhanced by aminotriazole, but strongly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) by azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU and moderately by Superoxide dismutase (SOD) and by deferoxamine. LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken together, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diphenylene iodonium), MPO (inhibitable by sodium azide), H 2 O 2 probably of intracellular origin (inhibitable by DMTU but not by catalase), and trace metals that contaminate salt solutions. The nature of the salt solutions employed to measure CL in activated PMNs is critical.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44510/1/10753_2004_Article_BF00918987.pd

Dhb Microcystins Discovered in USA Using an Online Concentration LC–MS/MS Platform

Based on current structural and statistical calculations, thousands of microcystins (MCs) can exist; yet, to date, only 246 MCs have been identified and only 12 commercial MC standards are available. Standard mass spectrometry workflows for known and unknown MCs need to be developed and validated for basic and applied harmful algal bloom research to advance. Our investigation focuses on samples taken in the spring of 2018 from an impoundment fed by Oser and Bischoff Reservoirs, Indiana, United States of America (USA). The dominant cyanobacterium found during sampling was Planktothrix agardhii. The goal of our study was to identify and quantify the MCs in the impoundment sample using chemical derivatization and mass spectrometry. Modifying these techniques to use online concentration liquid chromatography tandem mass spectrometry (LC&ndash;MS/MS), two untargeted MCs have been identified, [d-Asp3, Dhb7]-MC-LR and tentative [Dhb7]-MC-YR. [Dhb7]-MC-YR is not yet reported in the literature to date, and this was the first reported incidence of Dhb MCs in the United States. Furthermore, it was discovered that the commercially available [d-Asp3]-MC-RR standard was [d-Asp3, Dhb7]-MC-RR. This study highlights a workflow utilizing online concentration LC&ndash;MS/MS, high-resolution MS (HRMS), and chemical derivatization to identify isobaric MCs

Comparative Analysis of Microcystin Prevalence in Michigan Lakes by Online Concentration LC/MS/MS and ELISA

Fast and reliable workflows are needed to quantitate microcystins (MCs), a ubiquitous class of hepatotoxic cyanotoxins, so that the impact of human and environmental exposure is assessed quickly and minimized. Our goal was to develop a high-throughput online concentration liquid chromatography tandem mass spectrometry (LC/MS/MS) workflow to quantitate the 12 commercially available MCs and nodularin in surface and drinking waters. The method run time was 8.5 min with detection limits in the low ng/L range and minimum reporting levels between 5 and 10 ng/L. This workflow was benchmarked by determining the prevalence of MCs and comparing the Adda-ELISA quantitation to our new workflow from 122 samples representing 31 waterbodies throughout Michigan. The frequency of MC occurrence was MC-LA &gt; LR &gt; RR &gt; D-Asp3-LR &gt; YR &gt; HilR &gt; WR &gt; D-Asp3-RR &gt; HtyR &gt; LY = LW = LF, while MC-RR had the highest concentrations. MCs were detected in 33 samples and 13 of these samples had more than 20% of their total MC concentration from MCs not present in US Environmental Protection Agency (US EPA) Method 544. Furthermore, seasonal deviations between the LC/MS/MS and Adda-ELISA data suggest Adda-ELISA cross-reacts with MC degradation products. This workflow provides less than 24-h turnaround for quantification and also identified key differences between LC/MS/MS and ELISA quantitation that should be investigated further