A sensitized ENU mutagenesis screen for dominant genetic modifiers of thrombosis in the factor V Leiden mouse

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106086/1/jth05193.pd

The factor V Leiden mutation results in the in vivo loss of a critical FV anticoagulant function

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106106/1/jth05190.pd

Treatment of experimental adjuvant arthritis with a novel folate receptor-targeted folic acid-aminopterin conjugate

Introduction Folate receptor (FR)-expressing macrophages have been shown to accumulate at sites of inflammation, where they promote development of inflammatory symptoms. To target such a macrophage population, we designed and evaluated the biologic activity of EC0746, a novel folic acid conjugate of the highly potent antifolate, aminopterin. Methods Using a FR-positive subclone of murine macrophage-derived RAW264.7 cells and rat thioglycollate-elicited macrophages, we studied the effect of EC0746 on dihydrofolate reductase activity, cell proliferation, and cellular response towards bacterial lipopolysaccharide as well as IFNγ activation. The EC0746 anti-inflammatory activity, pharmacokinetics, and toxicity were also evaluated in normal rats or in rats with adjuvant-induced arthritis; that is, a FR-positive macrophage model that closely resembles rheumatoid arthritis in humans. Results EC0746 suppresses the proliferation of RAW264.7 cells and prevents the ability of nonproliferating rat macrophages to respond to inflammatory stimuli. In the macrophage-rich rat arthritis model, brief treatment with subcutaneously administered EC0746 is shown to mediate an FR-specific anti-inflammatory response that is more potent than either orally administered methotrexate or subcutaneously delivered etanercept. More importantly, EC0746 therapy is also shown to be ~40-fold less toxic than unmodified aminopterin, with fewer bone marrow and gastrointestinal problems. Conclusions EC0746 is the first high FR-binding dihydrofolate reductase inhibitor that demonstrates FR-specific anti-inflammatory activities both in vitro and in vivo. Our data reveal that a relatively toxic anti-inflammatory drug, such as aminopterin, can be targeted with folic acid to inflammatory macrophages and thereby relieve inflammatory symptoms with greatly reduced toxicity

Temporal evolution of shallow marine diagenetic environments: Insights from carbonate concretions

Early diagenesis of marine organic matter dramatically impacts Earth's surface chemistry by changing the burial potential of carbon and promoting the formation of authigenic mineral phases including carbonate concretions. Marine sediment-hosted carbonate concretions tend to form as a result of microbial anaerobic diagenetic reactions that degrade organic matter and methane, some of which require an external oxidant. Thus, temporal changes in the oxidation state of Earth's oceans may impart a first-order control on concretion authigenesis mechanisms through time. Statistically significant variability in concretion carbonate carbon isotope compositions indicates changes in shallow marine sediment diagenesis associated with Earth's evolving redox landscape. This variability manifests itself as an expansion in carbon isotope composition range broadly characterized by an increase in maximum and decrease in minimum isotope values through time. Reaction transport modelling helps to constrain the potential impacts of shifting redox chemistry and highlights the importance of organic carbon delivery to the seafloor, marine sulfate concentrations, methane production and external methane influx. The first appearance of conclusively anaerobic oxidation of methane-derived concretions occurs in the Carboniferous and coincides with a Paleozoic rise in marine sulfate. The muted variability recognized in older concretions (and in particular for Precambrian concretions) likely reflects impacts of a smaller marine sulfate reservoir and perhaps elevated marine dissolved inorganic carbon concentrations. Causes of the increase in carbon isotope maximum values through time are more confounding, but may be related to isotopic equilibration of dissolved inorganic carbon with externally derived methane. Ultimately the concretion isotope record in part reflects changes in organic matter availability and marine oxidation state, highlighting connections with the subsurface biosphere and diagenesis throughout geologic time

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

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

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

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

The Economic, Social and Administrative Pharmacy (ESAP) Discipline in US Schools and Colleges of Pharmacy

Schools and colleges of pharmacy in the United States have struggled over the past several decades with identifying a consistent title for the broad body of knowledge related to the social, economic, behavioral, and administrative aspects of pharmacy. This paper examines the educational background and professional experience of those teaching Economic, Social, Administrative Pharmacy (ESAP) content in the United States, to identify the type, frequency, and extent to which ESAP courses are taught in United State’s schools and colleges of pharmacy curricula, and to investigate the perceived importance of ESAP content among ESAP faculty. The data was collected by using a 23-item questionnaire was sent via email to 225 ESAP pharmacy faculties in the United States. 96 were returned completed for a 48 percent response rate. Most respondents are not housed in a standalone ESAP department, but would like to be. Both the educational backgrounds of ESAP faculty and the content taught within the discipline vary greatly. This may be because the content within the ESAP discipline is extremely broad. In addition, the diversification in content may be a result of both the diversity of instructor’s educational backgrounds and the lack of a generally approved definition for the ESAP field