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.     

Lumenal transmission of decapentaplegic in Drosophila imaginal discs

Drosophila imaginal discs are sac-like appendage primordia comprising apposed peripodial and columnar cell layers. Cell survival in disc columnar epithelia requires the secreted signal Decapentaplegic (DPP), which also acts as a gradient morphogen during pattern formation. The distribution mechanism by which secreted DPP mediates global cell survival and graded patterning is poorly understood. Here we report detection of DPP in the lumenal cavity between apposed peripodial and columnar cell layers of both wing and eye discs. We show that peripodial cell survival hinges upon DPP signal reception and implicate DPP-dependent viability of the peripodial epithelium in growth of the entire disc. These results are consistent with lumenal transmission of the DPP survival signal during imaginal disc development.     

Induction of cardiac cytochrome p450 in cocaine-treated mice

Cytochrome P450 (P450) is a ubiquitous family of enzymes responsible for the metabolism of a wide variety of drugs and their metabolites, including cocaine. To investigate the effects of cocaine on myocardial injuries and cardiac P450 expression, BALB/c mice were injected daily intraperitoneally with cocaine (30 mg/kg) or cocaine plus pretreatment of P450 inhibitors for 14 days. Tumor necrosis factor-alpha (TNF-alpha) content and creatine phosphokinase (CPK) activity in mice hearts and serums were significantly increased after long-term treatment with cocaine. Pretreatment with the P450 inhibitor, cimetidine (Cime, 50 mg/kg) or metyrapone (Mety, 40 mg/kg) abolished or significantly attenuated the effects of cocaine on TNF-alpha and CPK activity. Western blot analysis shows that mouse cardiac tissues express the P450 isoforms CYP1A1, CYP1A2, and CYP2J2. The protein levels normalized with cyclophilin A were 1.20 plus minus 0.07, 0.67 plus minus 0.03, and 1.48 plus minus 0.01 for CYP1A1, CYP1A2, and CYP 2J2, respectively. After cocaine administration, CYP2J2 increased by 43.6% and CYP1A1 increased by 108.5%, but CYP1A2 was not significantly altered. However, the cytochrome P450 inhibitors Cime and Mety suppressed the cocaine-induced increase in CYP1A1 and CYP2J2 expression. Moreover, application of Cime or Mety alone did not alter the level of cardiac TNF-alpha or the expression of P450. Our results demonstrate that long-term exposure to cocaine causes an increase in cardiac CYP1A1 and CYP2J2 concentration. We speculate that induction of P450 isoforms may cause cardiac injury due to cocaine metabolites locally catalyzed by P450 or the increase in P450 expression itself.