Biomedical subjects

John C Thompson

Publications and source records attributed to John C Thompson.

6 recordsLinked to original sources

PubMed2005

Smad1 and Smad8 function similarly in mammalian central nervous system development.

Smads 1, 5, and 8 are the intracellular mediators for the bone morphogenetic proteins (BMPs), which play crucial roles during mammalian development. Previous research has shown that Smad1 is important in the formation of the allantois, while Smad5 has been shown to be critical in the process of angiogenesis. To further analyze the BMP-responsive Smads, we disrupted the murine Smad8 gene utilizing the Cre/loxP system. A Smad8 hypomorphic allele (Smad8(Deltaexon3)) was constructed that contains an in-frame deletion of exon 3, removing one-third of the MH2 domain and a small portion of the linker region. Xenopus injection assays indicated that this Smad8 deletion allele is still functional but has reduced ventralizing capability compared to the wild type. Although Smad8(Deltaexon3/Deltaexon3) embryos are phenotypically normal, homozygotes of another hypomorphic allele of Smad8 (Smad8(3loxP)) containing a neomycin cassette within intron 3, phenocopy an embryonic brain defect observed in roughly 22% of Smad1(+/)(-) embryos analyzed at embryonic day 11.5. These observations suggest that BMP-responsive Smads have critical functions in the development of the mammalian central nervous system.

Alleles↗

PubMed2004

Smad2 and Smad3 coordinately regulate craniofacial and endodermal development.

Ligands of the transforming growth factor-beta (TGF-beta) superfamily are involved in numerous developmental and disease processes. TGF-beta, activins, and nodal ligands operate through the highly homologous Smad2 and Smad3 intracellular mediators. Smad2 mutants exhibit early embryonic lethality, while Smad3 mutants are viable, but show a plethora of postnatal phenotypes, including immune dysfunction and skeletal abnormalities. Previously, we have shown that the Smad2 and Smad3 genes function cooperatively during liver morphogenesis. Here we show that Smad2 and Smad3 are required at a full dosage for normal embryonic development. Animals lacking one allele of each gene exhibit a variably penetrant phenotype in which structures in the anterior and ventral midline are reduced or lost; additionally, we demonstrate that this craniofacial defect and the previously reported hepatic phenotypes are both due to defects in the definitive endoderm. A reduction of endodermal gene expression as well as a failure to displace the visceral endoderm occurs despite the formation of a normal foregut pocket. This precedes any defects in anterior patterning and likely causes the abnormalities observed in craniofacial and midline development, as well as hepatogenesis.

Animals↗

PubMed2004

Generation of novel conditional and hypomorphic alleles of the Smad2 gene.

Smad2 is an intracellular mediator of the transforming growth factor beta signaling (TGFbeta) pathway. It has been previously shown that, in the mouse, ablation of functional Smad2 results in embryonic lethality due to gastrulation defects. To circumvent the early lethality and study the spatially and temporally specific functions of Smad2, we utilized the Cre-loxP system to generate a Smad2 conditional allele. Here we show that a conditional allele, Smad2(flox), was generated. In this allele, exons 9 and 10 are flanked by loxP sites and the gene is functionally wildtype. Cre-mediated recombination results in a deletion allele which phenocopies our previously reported Smad2(DeltaC) null mutation. To generate this conditional allele, we first made a targeted mutation which introduced a floxed neo cassette into intron 10. This allele (Smad2(3loxP)) functions hypomorphically when placed opposite a null allele, and unlike the other published Smad2 hypomorphic allele, can be maintained in the homozygous state.

Alleles↗

PubMed2003

Extra-embryonic function of Rb is essential for embryonic development and viability.

The retinoblastoma (Rb) gene was the first tumour suppressor identified. Inactivation of Rb in mice results in unscheduled cell proliferation, apoptosis and widespread developmental defects, leading to embryonic death by day 14.5 (refs 2-4). However, the actual cause of the embryonic lethality has not been fully investigated. Here we show that loss of Rb leads to excessive proliferation of trophoblast cells and a severe disruption of the normal labyrinth architecture in the placenta. This is accompanied by a decrease in vascularization and a reduction in placental transport function. We used two complementary techniques-tetraploid aggregation and conditional knockout strategies-to demonstrate that Rb-deficient embryos supplied with a wild-type placenta can be carried to term, but die soon after birth. Most of the neurological and erythroid abnormalities thought to be responsible for the embryonic lethality of Rb-null animals were virtually absent in rescued Rb-null pups. These findings identify and define a key function of Rb in extra-embryonic cell lineages that is required for embryonic development and viability, and provide a mechanism for the cell autonomous versus non-cell autonomous roles of Rb in development.

Animals↗

PubMed2003

Clinical performance characteristics of a new photometric lithium assay: a multicenter study.

BACKGROUND: Therapeutic monitoring of lithium is important because of its narrow therapeutic range and therapeutic index, low protein binding and single route of elimination. We characterized a new photometric method that avoids the specialized requirements of ion-specific electrode (ISE), atomic absorption and flame emission methods. METHODS: Minimum detectable concentration (MDC), linearity and calibration drift over 65 days were determined. Within-run, between-run and total imprecision were assessed over 20 days in accordance with NCCLS EP5. Interference studies were conducted for 46 endogenous and exogenous compounds. Two production lots of the new photometric method (LI) were compared on the Dimension(R) RxL system and two ISE methods [Ciba Corning (n=124) and DuPont (n=131)], an established photometric method (Vitros) 950 system; n=63) and atomic absorption (Thermo-Jerell Ash; n=63). RESULTS: The MDC was 0.04 mmol/l. Linearity was demonstrated from 0.12 to 5.8 mmol/l by the regression equation: observed=(1.01 x expected)-0.0005 mmol/l, S(y/x)=0.03 mmol/l, r=0.999. Drift for the lithium calibrators over the 65-day study period was <5%, except for the lowest calibrator, which showed 0.04 mmol/l drift. None of the 46 potential interfering substances showed greater than a 6.5% difference between control and test solutions. ISE method comparisons showed the following: LI=(1.08 x Ciba Corning ISE)-0.15 mmol/l, S(y/x)=0.05, r=0.999, and LI=(1.03 x DuPont ISE)+0.00 mmol/l, S(y/x)=0.06 mmol/l, r=0.999. Comparison of the LI method with the atomic absorption and Vitros system showed proportionality error <10%. Bias between the LI method and atomic absorption was 7%, substantially less than that documented in proficiency surveys for the Vitros and other systems. No lot-to-lot or site-to-site differences were observed. CONCLUSION: This new photometric method is an attractive alternative for Li measurement and is adaptable to instruments having spectrophotometric capability.

Binding, Competitive↗

PubMed2003

Development of an automated photometric assay for serum lithium and use of binding equilibrium expressions to optimize results.

BACKGROUND: A spectrophotometric serum lithium assay and relevant reaction equilibria used in the assay development are described. METHODS: The dye, 7-nitro-2,12-dicarboxyl-16,17-dihydro-5H,15H-dibenzo[b,i] [1,11,4,5,7,8]dioxatetraazacyclotetradecine reacts rapidly with Li(+) in alkaline water/dimethylsulfoxide with an absorbance change. Sodium binds weakly, thus presenting potential inaccuracy from varying endogenous sodium. To minimize this problem, excess sodium ion was added from reagents. This diminishes the signal from lithium binding, thus lowering sensitivity, requiring selection of reagent sodium concentration for proper balance of performance characteristics. Absorptivities and binding constants were determined. Description of competing equilibria by an explicit cubic equation was used to predict sensitivity and effects of varying endogenous Na(+) concentration as functions of added NaCl. RESULTS: Concentration-absorbance data were satisfactorily predicted by the cubic equation, equilibrium constants, and absorptivities. Li(+) binds 300 times more tightly than Na(+). Interference was minimized, while sufficient sensitivity for needed precision was retained. A full evaluation is summarized separately in this issue. CONCLUSIONS: Explicit description of the competitive binding was successfully used to develop an automated liquid reagent assay for Li(+) with acceptable accuracy and precision.

Automation↗