Small-molecule cyclic alpha V beta 3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion

Abnormal adhesion of sickle red blood cells (SS RBCs) to vascular endothelium may play an important role in vasoocclusion in sickle cell disease. Accruing evidence shows that endothelial alpha V beta 3-integrin has an important role in SS RBC adhesion because of its ability to bind several adhesive proteins implicated in this interaction. In the present studies, we tested therapeutic efficacy of small-molecule cyclic pentapeptides for their ability to block alpha V beta 3-mediated SS RBC adhesion by using two well-established assay systems, i.e., cultured human umbilical vein endothelial cells (HUVEC) and artificially perfused mesocecum vasculature of the rat under flow conditions. We tested the efficacy of two RGD-containing cyclic pentapeptides, i.e., cRGDFV (EMD 66203) and cRGDF-ACHA (alpha-amino cyclohexyl carboxylic acid) (EMD 270179), based on their known ability to bind alpha V beta 3. An inactive peptide, EMD 135981 (cR beta-ADFV) was used as control. Cyclization and the introduction of D-Phe (F) results in a marked increase in the ability of cyclic peptides to selectively bind alpha V beta 3 receptors. In the mesocecum vasculature, both EMD 66203 and EMD 270179 ameliorated platelet-activating factor-induced enhanced SS RBC adhesion, postcapillary blockage, and significantly improved hemodynamic behavior. Infusion of a fluorescent derivative of EMD 66203 resulted in colocalization of the antagonist with vascular endothelium. Also, pretreatment of HUVEC with either alpha V beta 3 antagonist resulted in a significant decrease in SS RBC adhesion. Because of their metabolic stability, the use of these cyclic alpha V beta 3 antagonists may constitute a novel therapeutic strategy to block SS RBC adhesion and associated vasoocclusion under flow conditions.     

An unconventional human Ccr4-Caf1 deadenylase complex in nuclear cajal bodies

mRNA deadenylation is a key process in the regulation of translation and mRNA turnover. In Saccharomyces cerevisiae, deadenylation is primarily carried out by the Ccr4p and Caf1p/Pop2p subunits of the Ccr4-Not complex, which is conserved in eukaryotes including humans. Here we have identified an unconventional human Ccr4-Caf1 complex containing hCcr4d and hCaf1z, distant human homologs of yeast Ccr4p and Caf1p/Pop2p, respectively. The hCcr4d-hCaf1z complex differs from conventional Ccr4-Not deadenylase complexes, because (i) hCaf1z and hCcr4d concentrate in nuclear Cajal bodies and shuttle between the nucleus and cytoplasm and (ii) the hCaf1z subunit, in addition to rapid deadenylation, subjects substrate RNAs to slow exonucleolytic degradation from the 3' end in vitro. Exogenously expressed hCaf1z shows both of those activities on reporter mRNAs in human HeLa cells and stimulates general mRNA decay when restricted to the cytoplasm by deletion of its nuclear localization signal. These observations suggest that the hCcr4d-hCaf1z complex may function either in the nucleus or in the cytoplasm after its nuclear export, to degrade polyadenylated RNAs, such as mRNAs, pre-mRNAs, or those RNAs that are polyadenylated prior to their degradation in the nucleus.     

Redox activity associated with the maturation and capacitation of mammalian spermatozoa

As rat spermatozoa undergo epididymal maturation, they acquire the ability to exhibit a spontaneous burst of luminol-peroxidase-dependent chemiluminescence when released into a simple, defined culture medium. This activity was suppressed by inhibitors of plasma membrane redox systems such as diphenylene iodonium, p-chloromercuribenzenesulfonic acid, and capsaicin, but was resistant to inhibition by resiniferatoxin and rotenone. The luminol-peroxidase signal was dependent on the presence of bicarbonate, enhanced by the substitution of fructose for glucose, and severely suppressed by desferoxamine, superoxide dimutase, and catalase. Both L- and D-arginine were stimulatory, suggesting the involvement of *NO in this spontaneous chemiluminescence activity. The L-arginine-dependent, but not the D-arginine-dependent, activity was significantly suppressed by an inhibitor of nitric oxide synthase (N(G)-nitro-L-arginine methyl ester). L- and D-arginine could also stimulate redox activity observed in immature caput epididymal cells, but only after prolonged incubation. The inhibitory effects of uric acid and ascorbate suggested the chemiluminescence signal might be induced by peroxynitrite. This conclusion was supported by confocal imaging of the cells following treatment with 4-amino-5-methylamino-2',7'-difluorofluorescein. Stimulation or suppression of the redox activity detected by luminol-peroxidase led to corresponding changes in the ability of the spermatozoa to exhibit acrosomal exocytosis, indicating that this pathway is of fundamental biological significance.     

The porphobilinogen synthase catalyzed reaction mechanism

Porphobilinogen synthase (PBGS) catalyzes the first common reaction in the biosynthesis of the tetrapyrroles, the asymmetric condensation of two molecules of delta-aminolevulinic acid to form porphobilinogen. There is a variable requirement for an essential active site zinc that necessitates consideration of PBGS as an enzyme that may exhibit phylogenetic diversity in its chemical reaction mechanism. Recent crystal structures suggest reaction mechanisms that involve two covalent Schiff base linkages between adjacent active site lysine residues and each of the two substrate molecules. The reaction appears to stall at a covalently bound almost-product intermediate that is poised for breakdown to product upon binding of a substrate molecule to an adjacent active site and a subsequent conformational change.     

A novel proteomic coculture model of prostate cancer cell growth

Chemotherapy and androgen ablation therapy are only temporarily effective against prostate cancer, and current studies are ongoing to test agents that target proteins responsible for autocrine and paracrine stimulated growth. Given limitations of current laboratory models to test the effect of these agents on cell growth and protein targets, we developed a coculture model that can distinguish paracrine stimulated growth and effects on proteins. We found that LNCaP prostate cancer cells and an immortalized rat prostate cell line transfected to overexpress the antiapoptotic resistance protein Bcl-2 were stimulated to grow (>2-fold increase, p < 0.01) through autocrine effects from additional cells in an upper chamber of our system. Using a proteomic approach with a two-dimensional differential in gel electrophoresis method to increase fidelity, four proteins were found to increase after autocrine induced growth stimulation. These proteins were all identified by mass spectrometry as enzymes in the glycolytic pathway, validating the ability of this system to detect both clonogenic growth and the effect on proteins. These data, therefore, demonstrate a novel coculture model for further study of agents that target proteins in pathways of paracrine or autocrine stimulated cell growth.     

Chemical complementation: small-molecule-based genetic selection in yeast

Protein and metabolic engineering would greatly benefit from a general system linking the presence of a small molecule to the power of genetic selection. We use nuclear receptors to link the survival of Saccharomyces cerevisiae to the presence of small molecules through genetic selection, extending classical genetic complementation to a new "chemical complementation." In this system the Gal4 DNA-binding domain is fused to ligand-binding domains from two nuclear receptors, expressed in the strain PJ69-4A, and grown on plates containing known ligands for the receptors. Yeast survive on selective plates only in the presence of a nuclear receptor and the corresponding ligand. Mutagenesis can increase the sensitivity of chemical complementation. This system may be extended to engineer nuclear receptors for practically any small molecule through directed evolution coupled to genetic selection, and for performing metabolic engineering in yeast.     

Enhanced selection for MHC diversity in social tuco-tucos

To explore the effects of behavior and demography on balancing selection at major histocompatibility complex (MHC) loci, we examined allelic diversity at exon 2 of the MHC class II DQbeta locus in a social and a solitary species of tuco-tuco (Rodentia: Ctenomyidae: Ctenomys), both of which occur in the same valley in southwestern Argentina. By comparing patterns of diversity at this MHC gene to the diversity evident at fifteen microsatellite loci, we demonstrate that balancing selection at the DQbeta locus is enhanced in the social species compared to its solitary congener. These findings have intriguing implications for the role of behavioral and demographic parameters in maintaining diversity at MHC loci.     

Clathrin-protein interactions

There is a complex network of protein–protein and protein–lipid interactions that underlie clathrin-mediated vesicular traffic in all compartmentalized cells from yeast to man. Major progress has been made in the determination of the three-dimensional structures of many of the components. Recently, there has been an explosion in the identification and characterization of clathrin binding partners. This review integrates the structural and biochemical information that is currently available to present a unified view of how many clathrin binding partners interact with clathrin.