Nogo on the go

Growth inhibition in the central nervous system (CNS) is a major barrier to axon regeneration. Recent findings indicate that three distinct myelin proteins, myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte-myelin glycoprotein (OMgp), inhibit axon growth by binding a common receptor, the Nogo66 receptor (NgR), and likely converge on a common signaling cascade.     

In addition to the SH3 binding region, multiple regions within the N-terminal noncatalytic portion of the cAMP-specific phosphodiesterase, PDE4A5, contribute to its intracellular targeting

The long cyclic AMP (cAMP)-specific phosphodiesterase isoform, PDE4A5 (PDE4A subfamily isoform variant 5), when transiently expressed in COS-7 cells, was shown in subcellular fractionation studies to be associated with both membrane and cytosol fractions, with immunofluorescence analyses identifying PDE4A5 as associated both with ruffles at the cell margin and also at a distinct perinuclear localisation. Deletion of the first nine amino acids of PDE4A5 (1) ablated its ability to interact with the SH3 domain of the tyrosyl kinase, LYN; (2) reduced, but did not ablate, membrane association; and (3) disrupted the focus of PDE4A5 localisation within ruffles at the cell margin. This deleted region contained a Class I SH3 binding motif of similar sequence to those identified by screening a phage display library with the LYN-SH3 domain. Truncation to remove the PDE4A5 isoform-specific N-terminal region caused a further reduction in membrane association and ablated localisation at the cell margin. Progressive truncation to delete the PDE4A long isoform common region and then the long isoform-specific UCR1 did not cause any further change in membrane association or intracellular distribution. However, deletion up to the super-short form splice junction generated an entirely soluble 'core' PDE4A species. We propose that multiple sites in the N-terminal noncatalytic portion of PDE4A5 have the potential to associate with intracellular structures and thus define its intracellular localisation. At least two such sites lie within the PDE4A5 isoform-specific N-terminal region and these appear to be primarily responsible for targeting PDE4A5 to, and organising it within, the cell margin; one is an SH3 binding motif able to interact with LYN kinase and the other lies within the C-terminal portion of the PDE4A5 unique region. A third membrane association region is located within the N-terminal portion of UCR2 and appears to be primarily responsible for targeting to the perinuclear region. Progressive N-terminal truncation, to delete defined regions of PDE4A5, identified activity changes occurring upon deletion of the SH3 binding site region and then upon deletion of the membrane association site region located within UCR2. This suggests that certain of these anchor sites may not only determine intracellular targeting but may also transduce regulatory effects on PDE4A5 activity.     

The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism

The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants; however, no conditions suitable for the unambiguous assay of the enzyme are known. As a result, all attempts to purify the protein and clone its corresponding gene have failed. By screening for plants that accumulate reduced levels of soluble fluorescent phenylpropanoid secondary metabolites, we have identified a number of Arabidopsis mutants that display a reduced epidermal fluorescence (ref) phenotype. Using radiotracer-feeding experiments, we have determined that the ref8 mutant is unable to synthesize caffeic acid, suggesting that the mutant is defective in a gene required for the activity or expression of C3H. We have isolated the REF8 gene using positional cloning methods, and have verified that it encodes C3H by expression of the wild-type gene in yeast. Although many previous reports in the literature have suggested that C3H is a phenolase, the isolation of the REF8 gene demonstrates that the enzyme is actually a cytochrome P450-dependent monooxygenase. Although the enzyme accepts p-coumarate as a substrate, it also exhibits significant activity towards other p-hydroxylated substrates. These data may explain the previous difficulties in identifying C3H activity in plant extracts and they indicate that the currently accepted version of the lignin biosynthetic pathway is likely to be incorrect.     

Stereoselective and improved syntheses and anticancer testing of 3'-O-silatranylthymidines

A stereoselective synthesis of 3′-O-((R,R,R)-trimethylsilatranyl)thymidine (R,R,R-1) and synthesis of 3′-O-silatranylthymidine (5) via an improved silatranylation procedure using tetrakis(dimethylamino)silane are reported. Diastereomeric mixture 1 showed more activity than R,R,R-1 or 5 in a primary anticancer screen against breast, CNS, and lung cell lines; demonstrating the import of the configuration and presence, respectively, of the silatrane methyl groups for growth inhibition.     

Toll-like receptors: a family of pattern-recognition receptors in mammals

The innate immune system uses a variety of germline-encoded pattern-recognition receptors that recognize conserved microbial structures or pathogen-associated molecular patterns, such as those that occur in the bacterial cell-wall components peptidoglycan and lipopolysaccharide. Recent studies have highlighted the importance of Toll-like receptors (TLRs) as a family of pattern-recognition receptors in mammals that can discriminate between chemically diverse classes of microbial products. First identified on the basis of sequence similarity with the Drosophila protein Toll, TLRs are members of an ancient superfamily of proteins, which includes related proteins in invertebrates and plants. TLRs activate innate immune defense reactions, such as the release of inflammatory cytokines, but increasing evidence supports an additional critical role for TLRs in orchestrating the development of adaptive immune responses. The sequence similarity between the intracellular domains of the TLRs and the mammalian interleukin-1 and interleukin-18 cytokine receptors reflects the use of a common intracellular signal-transduction cascade triggered by these receptor classes. But more recent findings have demonstrated that there are in fact TLR-specific signaling pathways and cellular responses. Thus, TLRs function as sentinels of the mammalian immune system that can discriminate between diverse pathogen-associated molecular patterns and then elicit pathogen-specific cellular immune responses.     

Software and methods for oligonucleotide and cDNA array data analysis

Two HTML-based programs were developed to analyze and filter gene-expression data: 'Bullfrog' for Affymetrix oligonucleotide arrays and 'Spot' for custom cDNA arrays. The programs provide intuitive data-filtering tools through an easy-to-use interface. A background subtraction and normalization program for cDNA arrays was also built that provides an informative summary report with data-quality assessments. These programs are freeware to aid in the analysis of gene-expression results and facilitate the search for genes responsible for interesting biological processes and phenotypes.     

Proteasome subunit Rpn1 binds ubiquitin-like protein domains

The yeast protein Rad23 belongs to a diverse family of proteins that contain an amino-terminal ubiquitin-like (UBL) domain. This domain mediates the binding of Rad23 to proteasomes, which in turn promotes DNA repair and modulates protein degradation, possibly by delivering ubiquitinylated cargo to proteasomes. Here we show that Rad23 binds proteasomes by directly interacting with the base subcomplex of the regulatory particle of the proteasome. A component of the base, Rpn1, specifically recognizes the UBL domain of Rad23 through its leucine-rich-repeat-like (LRR-like) domain. A second UBL protein, Dsk2, competes with Rad23 for proteasome binding, which suggests that the LRR-like domain of Rpn1 may participate in the recognition of several ligands of the proteasome. We propose that the LRR domain of Rpn1 may be positioned in the base to allow the cargo proteins carried by Rad23 to be presented to the proteasomal ATPases for unfolding. We also report that, contrary to expectation, the base subunit Rpn10 does not mediate the binding of UBL proteins to the proteasome in yeast, although it can apparently contribute to the binding of ubiquitin chains by intact proteasomes.     

Crystal doping aided by rapid expansion of supercritical solutions

The purpose of this study was to test the utility of rapid expansion of supercritical solution (RESS) based cocrystallizations in inducing polymorph conversion and crystal disruption of chlorpropamide (CPD). CPD crystals were recrystallized by the RESS process utilizing supercritical carbon dioxide as the solvent. The supercritical region investigated for solute extraction ranged from 45 to 100°C and 2000 to 8000 psi. While pure solute recrystallization formed stage I of these studies, stage II involved recrystallization of CPD in the presence of urea (model impurity). The composition, morphology, and crystallinity of the particles thus produced were characterized utilizing techniques such as microscopy, thermal analysis, x-ray powder diffractometry, and high-performance liquid chromatography. Also, comparative evaluation between RESS and evaporative crystallization from liquid solvents was performed. RESS recrystallizations of commercially available CPD (form A) resulted in polymorph conversion to metastable forms C and V, depending on the temperature and pressure of the recrystallizing solvent. Cocrystallization studies revealed the formation of eutectic mixtures and solid solutions of CPD+urea. Formation of the solid solutions resulted in the crystal disruption of CPD and subsequent amorphous conversion at urea levels higher than 40% wt/wt. Consistent with these results were the reductions in melting point (up to 9°C) and in the ΔH_fvalues of CPD (up to 50%). Scanning electron microscopy revealed a particle size reduction of up to an order of magnitude upon RESS processing. Unlike RESS, recrystallizations from liquid organic solvents lacked the ability to affect polymorphic conversions. Also, the incorporation of urea into the lattice of CPD was found to be inadequate. In providing the ability to control both the particle and crystal morphologies of active pharmaceutical ingredients, RESS proved potentially advantageous to crystal engineering. Rapid crystallization kinetics were found vital in making RESS-based doping superior to conventional solvent-based cocrystallizations.