A dedicated flavin-dependent monooxygenase catalyzes the hydroxylation of demethoxyubiquinone into ubiquinone (coenzyme Q) in Arabidopsis

Ubiquinone (Coenzyme Q) is a vital respiratory cofactor and liposoluble antioxidant. In plants, it is not known how the C-6 hydroxylation of demethoxyubiquinone, the penultimate step in ubiquinone biosynthesis, is catalyzed. The combination of cross-species gene network modeling along with mining of embryo-defective mutant databases of Arabidopsis thaliana identified the embryo lethal locus EMB2421 (At1g24340) as a top candidate for the missing plant demethoxyubiquinone hydroxylase. In marked contrast with prototypical eukaryotic demethoxyubiquinone hydroxylases, the catalytic mechanism of which depends on a carboxylate-bridged di-iron domain, At1g24340 is homologous to FAD-dependent oxidoreductases that instead use NAD(P)H as an electron donor. Complementation assays in Saccharomyces cerevisiae and Escherichia coli demonstrated that At1g24340 encodes a functional demethoxyubiquinone hydroxylase and that the enzyme displays strict specificity for the C-6 position of the benzoquinone ring. Laser-scanning confocal microscopy also showed that GFP-tagged At1g24340 is targeted to mitochondria. Silencing of At1g24340 resulted in 40 to 74% decrease in ubiquinone content and de novo ubiquinone biosynthesis. Consistent with the role of At1g24340 as a benzenoid ring modification enzyme, this metabolic blockage could not be bypassed by supplementation with 4-hydroxybenzoate, the immediate precursor of ubiquinone''s ring. Unlike in yeast, in Arabidopsis overexpression of demethoxyubiquinone hydroxylase did not boost ubiquinone content. Phylogenetic reconstructions indicated that plant demethoxyubiquinone hydroxylase is most closely related to prokaryotic monooxygenases that act on halogenated aromatics and likely descends from an event of horizontal gene transfer between a green alga and a bacterium.     

An equine protozoal myeloencephalitis challenge model testing a second transport after inoculation with Sarcocystis neurona sporocysts

Previous challenge studies performed at Ohio State University involved a transport-stress model where the study animals were dosed with Sarcocystis neurona sporocysts on the day of arrival. This study was to test a second transportation of horses after oral inoculation with S. neurona sporocysts. Horses were assigned randomly to groups: group 1, transported 4 days after inoculation (DAI); group 2, at 11 DAI; group 3, at 18 DAI; and group 4, horses were not transported a second time (controls). An overall neurologic score was determined on the basis of a standard numbering system used by veterinarians. All scores are out of 5, which is the most severely affected animal. The mean score for the group 1 horses was 2.42; group 2 horses was 2.5; group 3 horses was 2.75; and group 4 horses was 3.25. Because the group 4 horses did not have a second transport, they were compared with all other groups. Statistically different scores were present between group 4 and groups 1 and 2. There was no difference in the time of seroconversion between groups. There was a difference between the time of onset of first clinical signs between groups 1 and 4. This difference was likely because of the different examination days. Differences in housing and handling were likely the reason for the differences in severity of clinical signs. This model results in consistent, significant clinical signs in all horses at approximately the same time period after inoculation but was most severe in horses that did not experience a second transport.     

Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening

Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins. To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine-induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases.     

The prodomain of BMP-7 targets the BMP-7 complex to the extracellular matrix

Biochemical and biophysical methods are used to show that BMP-7 is secreted as a stable complex consisting of the processed growth factor dimer noncovalently associated with its two prodomain propeptide chains and that the BMP-7 complex is structurally similar to the small transforming growth factor beta (TGFbeta) complex. Because the prodomain of TGFbeta interacts with latent TGFbeta-binding proteins, a family of molecules homologous to the fibrillins, the prodomain of BMP-7 was tested for binding to fibrillin-1 or to LTBP-1. The BMP-7 prodomain and BMP-7 complex, but not the separated growth factor dimer, interact with N-terminal regions of fibrillin-1. This interaction may target the BMP-7 complex to fibrillin microfibrils in the extracellular matrix. Immunolocalization of BMP-7 in tissues like the kidney capsule and skin reveals co-localization with fibrillin. However, BMP-7 immunolocalization in other tissues known to be active sites for BMP-7 signaling is not apparent, suggesting that immunolocalization of BMP-7 in certain tissues represents specific extracellular storage sites. These studies suggest that the prodomains of TGFbeta-like growth factors are important for positioning and concentrating growth factors in the extracellular matrix. In addition, they raise the possibility that prodomains of other TGFbeta-like growth factors interact with fibrillins and/or LTBPs and are also targeted to the extracellular matrix.     

Fine tuning of growth factor signals depends on fibrillin microfibril networks

Growth factors, potent regulators of cell differentiation, tissue morphogenesis, tissue homeostasis, and cellular response to injury, reside in the extracellular matrix. Genetic evidence in humans and mice as well as biochemical data implicate fibrillins and LTBPs in the extracellular control of TGFbeta and BMP signaling. Fibrillins and LTBPs form tissue-specific and temporally regulated microfibril networks. In the developing embryo, three fibrillins and four LTBPs contribute molecular heterogeneity to microfibril networks, and provide different templates upon which TGFbeta-related growth factors can be positioned. By accommodating this molecular heterogeneity, microfibril architecture can orchestrate a variety of different signals in very specific tissue locations. Human fibrillinopathies display a broad phenotypic spectrum from tall to short stature, from hypermobile joints to joint contractures and stiffness, and from severe to mild or no cardiovascular manifestations. A spectrum of growth factor dysregulation may be caused by differential effects of mutations in fibrillins on microfibril architecture, thus altering appropriate targeting or positioning of growth factors within microfibril networks. Growth factor dysregulation may help to explain the broad phenotypic spectrum of the fibrillinopathies.     

Developmentally regulated alternative splicing of the alpha1(XI) collagen chain: spatial and temporal segregation of isoforms in the cartilage of fetal rat long bones.

Type XI collagen is a component of the heterotypic collagen fibrils of fetal cartilage and is required to maintain the unusually thin diameter of these fibrils. The mature matrix form of the molecule retains an N-terminal variable region whose structure is modulated by alternative exon splicing that is tissue-specific and developmentally regulated. In the alpha1(XI) chain, antibodies to two of the peptides, p6b and p8, encoded by the alternatively spliced exons localized these epitopes to the surface of the collagen fibrils and were used to determine the pattern of isoform expression during the development of rat long bones (humerus). Expression of the p6b isoform was restricted to the periphery of the cartilage underlying the perichondrium of the diaphysis, a pattern that appears de novo at embryonic Day (E) 14. P8 isoforms appeared to be associated with early stages of chondrocyte differentiation and were detected throughout prechondrogenic mesenchyme and immature cartilage. After E16, p8 isoforms gradually disappeared from the diaphysis and then from the epiphysis preceding chondrocyte hypertrophy, but were highly evident at the periarticular joint surface, where ongoing chondrogenesis accompanies the formation of articular cartilage. The spatially restricted and differentiation-specific distribution of alpha1(XI) isoforms is evidence that Type XI collagen participates in skeletal development via a mechanism that may be distinct from regulation of fibrillogenesis.     

Severely impaired learning and altered neuronal morphology in mice lacking NMDA receptors in medium spiny neurons

The striatum is composed predominantly of medium spiny neurons (MSNs) that integrate excitatory, glutamatergic inputs from the cortex and thalamus, and modulatory dopaminergic inputs from the ventral midbrain to influence behavior. Glutamatergic activation of AMPA, NMDA, and metabotropic receptors on MSNs is important for striatal development and function, but the roles of each of these receptor classes remain incompletely understood. Signaling through NMDA-type glutamate receptors (NMDARs) in the striatum has been implicated in various motor and appetitive learning paradigms. In addition, signaling through NMDARs influences neuronal morphology, which could underlie their role in mediating learned behaviors. To study the role of NMDARs on MSNs in learning and in morphological development, we generated mice lacking the essential NR1 subunit, encoded by the Grin1 gene, selectively in MSNs. Although these knockout mice appear normal and display normal 24-hour locomotion, they have severe deficits in motor learning, operant conditioning and active avoidance. In addition, the MSNs from these knockout mice have smaller cell bodies and decreased dendritic length compared to littermate controls. We conclude that NMDAR signaling in MSNs is critical for normal MSN morphology and many forms of learning.     

Discovery and synthesis of novel 4-aminopyrrolopyrimidine Tie-2 kinase inhibitors for the treatment of solid tumors

The synthesis and biological evaluation of novel Tie-2 kinase inhibitors are presented. Based on the pyrrolopyrimidine chemotype, several new series are described, including the benzimidazole series by linking a benzimidazole to the C5-position of the 4-amino-pyrrolopyrimidine core and the ketophenyl series synthesized by incorporating a ketophenyl group to the C5-position. Medicinal chemistry efforts led to potent Tie-2 inhibitors. Compound 15, a ketophenyl pyrrolopyrimidine urea analog with improved physicochemical properties, demonstrated favorable in vitro attributes as well as dose responsive and robust oral tumor growth inhibition in animal models.