ABSTRACT
Acute renal failure (ARF) has high mortality and no effective treatment. Nitric oxide (NO) delivery represents a credible means of preventing the damaging effects of vasoconstriction, central to ARF, but design of drugs with the necessary renoselectivity is challenging. Here, we developed N-hydroxyguanidine NO donor drugs that were protected against spontaneous NO release by linkage to glutamyl adducts that could be cleaved by γ-glutamyl transpeptidase (γ-GT), found predominantly in renal tissue. Parent NO donor drug activity was optimized in advance of glutamyl adduct prodrug design. A lead compound that was a suitable substrate for γ-GT-mediated deprotection was identified. Metabolism of this prodrug to the active parent compound was confirmed in rat kidney homogenates, and the prodrug was shown to be an active vasodilator in rat isolated perfused kidneys (EC50 ~50 µM). The data confirm that glutamate protection of N-hydroxyguanidines is an approach that might hold promise in ARF.
Subject(s)
Acute Kidney Injury/drug therapy , Guanidines/pharmacology , Kidney/drug effects , Nitric Oxide Donors/pharmacology , Acute Kidney Injury/metabolism , Animals , Chromatography, High Pressure Liquid , Crystallography, X-Ray , Glutamic Acid/chemistry , Guanidines/chemical synthesis , Guanidines/metabolism , Hydroxylamines , In Vitro Techniques , Kidney/metabolism , Male , Models, Chemical , Molecular Structure , Nitric Oxide/metabolism , Nitric Oxide Donors/chemical synthesis , Nitric Oxide Donors/metabolism , Prodrugs/chemical synthesis , Prodrugs/metabolism , Prodrugs/pharmacology , Rats , Rats, Wistar , Vasodilator Agents/chemical synthesis , Vasodilator Agents/metabolism , Vasodilator Agents/pharmacologyABSTRACT
To gain global insights into the role of the well-known repressive splicing regulator PTB, we analyzed the consequences of PTB knockdown in HeLa cells using high-density oligonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB-repressed and PTB-activated exons showed a distinct arrangement of motifs with pyrimidine-rich motif enrichment within and upstream of repressed exons but downstream of activated exons. The N-terminal half of PTB was sufficient to activate splicing when recruited downstream of a PTB-activated exon. Moreover, insertion of an upstream pyrimidine tract was sufficient to convert a PTB-activated exon to a PTB-repressed exon. Our results show that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons.
Subject(s)
Alternative Splicing , Polypyrimidine Tract-Binding Protein/metabolism , Base Sequence , Exons , HeLa Cells , Humans , Introns , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Polypyrimidine Tract-Binding Protein/genetics , Silencer Elements, TranscriptionalABSTRACT
The Kruppel-like factor, KLF13, is a member of a family of transcription factors shown to be involved in haematopoietic development. Here we show that KLF13 is involved in the development of B and T cells at multiple stages. Expression of KLF13 in the thymus was maximal in the DP population and in KLF13(-/-) deficient mice there was an accumulation of DP thymocytes and reduction of CD4(+)SP cells. Cell-surface expression of CD3(high), CD8, CD5 and HSA were altered on KLF13(-/-) DP cells, consistent with a defect in TCR signalling and at the DP to SP transition in KLF13(-/-) mice. KLF13 is also expressed in peripheral T-cells and peripheral T cell activation was impaired in KLF13(-/-) mice. Analysis of early B cell development in the bone marrow (BM) revealed a partial arrest of B cells at the transition from CD43(+) to CD43(-) pre-B cell, a transition that requires signalling through the pre-BCR. The proportion of IgM(+)/IgD(+) mature B cells was also increased in the BM of the KLF13(-/-) mice. This finding is consistent with a reduction in the strength of BCR signal or an accumulation of recirculating B cells from the periphery. Analysis of splenocytes isolated from KLF13(-/-) mice revealed an increase in the expression of CD21 and CD23 on B220(+) B cells, demonstrating a negative regulatory role for KLF13 in co-regulation of expression of CD21 and CD23. Thus KLF13 is involved at multiple different checkpoints in development that require signalling through the TCR, pre-BCR or mature BCR.
Subject(s)
B-Lymphocytes/metabolism , Cell Cycle Proteins/metabolism , Kruppel-Like Transcription Factors/metabolism , Receptors, Antigen, T-Cell/metabolism , Repressor Proteins/metabolism , T-Lymphocyte Subsets/metabolism , Thymus Gland/metabolism , Animals , B-Lymphocytes/immunology , CD5 Antigens/metabolism , Cell Differentiation , Kruppel-Like Transcription Factors/deficiency , Lymphocyte Activation/physiology , Mice , Mice, Knockout , Receptors, Complement 3d/metabolism , Receptors, IgE/metabolism , T-Lymphocyte Subsets/immunologyABSTRACT
To study the function of the Krüppel-like transcription factor KLF13 in vivo, we generated mice with a disrupted Klf13 allele. Although Klf13(-/-) mice are viable, fewer mice were present at 3 weeks than predicted by Mendelian inheritance. Viable Klf13(-/-) mice had reduced numbers of circulating erythrocytes and a larger spleen. The spleen contained an increased number of Ter119(med)CD71(hi), Ter119(hi)CD71(hi), and Ter119(hi)CD71(med) cells but not Ter119(hi)CD71(-) cells, indicating an increase in less mature erythroblasts. A higher proportion of the Ter119(med)CD71(hi) cells were proliferating, indicating that the mice were under a degree of erythropoietic stress. These data indicate that KLF13 is involved in the normal control of erythropoiesis.
Subject(s)
Erythropoiesis , Kruppel-Like Transcription Factors/deficiency , Splenomegaly/physiopathology , Animals , Apoptosis/genetics , Base Sequence , Blood Cell Count , Bone Marrow/pathology , Cell Cycle Proteins/genetics , Cell Differentiation , Cell Proliferation , Erythroblasts/pathology , Gene Deletion , Gene Expression Regulation , Gene Targeting , Genotype , Kruppel-Like Transcription Factors/genetics , Mice , Mice, Knockout , Molecular Sequence Data , Organ Size , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repressor Proteins/genetics , Splenomegaly/pathologyABSTRACT
The use of reporter genes such as beta-galactosidase and green fluorescent protein is a powerful molecular tool for the visualization of in-vivo gene expression. In this paper, we describe the generation of mice that possess a beta-galactosidase reporter gene introduced into the endothelin-B receptor locus by homologous recombination in embryonic stem cells. These mice express beta- galactosidase wherever endothelin-B is expressed and therefore provide a precise in-vivo localization profile of endothelin-B expression.
