Microcephaly models in the developing zebrafish retinal neuroepithelium point to an underlying defect in metaphase progression

Autosomal recessive primary microcephaly (MCPH) is a congenital disorder characterized by significantly reduced brain size and mental retardation. Nine genes are currently known to be associated with the condition, all of which encode centrosomal or spindle pole proteins. MCPH is associated with a reduction in proliferation of neural progenitors during fetal development. The cellular mechanisms underlying the proliferation defect, however, are not fully understood. The zebrafish retinal neuroepithelium provides an ideal system to investigate this question. Mutant or morpholino-mediated knockdown of three known MCPH genes (stil, aspm and wdr62) and a fourth centrosomal gene, odf2, which is linked to several MCPH proteins, results in a marked reduction in head and eye size. Imaging studies reveal a dramatic rise in the fraction of proliferating cells in mitosis in all cases, and time-lapse microscopy points to a failure of progression through prometaphase. There was also increased apoptosis in all the MCPH models but this appears to be secondary to the mitotic defect as we frequently saw mitotically arrested cells disappear, and knocking down p53 apoptosis did not rescue the mitotic phenotype, either in whole retinas or clones.     

Synthetic,Structural, and Conformational Studies of Methylated Ring-Expanded Nucleosides Containing the Ihidazo[4, 5-e][1, 4]Diazepine Ring System

Abstract

Syntheses of 4- and 7-methyl 4, 5, 7, 8-tetrahydro-6H-3-(β-ribofuranosyl)imidazo[4, 5-e] [1, 4]diazepine-5, 8-dione, 3 and 1, respectively, are reported. Single-crystal X-ray diffraction analysis of the aglycon of 3 aided in confirming the site of methylation in 3, and that of 4 in elucidating the solid state conformation of 4. Solution conformations of 3 and 4, along with their parent nucleoside 1 and the latter's 1-glycosyl regioisomer 2, were investigated by NOE and CD measurements.     

The Synthesis of Novel Regioisomeric Ring-Expanded Xanthine Nucleosides Containing The 5:7-Fused Imidazo[4,5-e][1,2,4]Triazepine Ring System

Abstract

The syntheses of novel regioisomeric ring-expanded purine nucleosides containing the imidazo[4,5-e][1,2,4]triazepine nucleus are reported. The glycosylation of the heterocycle 3,4,6,7-tetrahydroimidazo[4,5-e][1,2,4]triazepine-5,8-dione (2a) by the stannic chloride procedure gave nucleosides 3 and 4, with the sugar moiety attached at the 7-and 3-positions of the heterocycle, respectively. On the other hand, the mercuric cyanide procedure for glycosylation of 2a yielded nucleosides 4 and 5, with the sugar attached at the 1-position in the latter. In either procedure, 4 was the minor isomer and was obtained only in trace amounts. While debenzoylation of 3 and 5 provided the respective parent nucleosides 8 and 10, that of 4 resulted in ring-opening to produce 9. Attempted enzymic glycosylation of 2a with purine nucleoside phosphorylase failed to yield any nucleoside product.     

The Synthesis,Structure, and Conformation of 2′-Deoxy Analogues of „Fat“ Xanthine Nucleosides,Containing the Imidazo[4,5-e][1,4]Diazepine Ring System

Abstract

The syntheses of 1-(2-deoxy-β-D-erythro-pentofuranosyl)-4,5,7,8-tetrahydro-6H-imidazo[4,5-e] [1,4]diazepine-5,8-dione (9β), its 3-glycosyl regioisomer (16β), and their respective α anomers (9α and 16α), are reported. Conformational and configurational studies, employing ¹H NMR NOE and CD spectroscopy, are described. The single-crystal X-ray structural analysis of 9β is presented. The attempted enzymic glycosylation of the heterocyclic base 6 with a bacterial purine nucleoside phosphorylase was not successful.     

Global Subcellular Characterization of Protein Degradation Using Quantitative Proteomics

Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ∼5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution.Targeted protein degradation is an important regulatory mechanism that allows co-ordination of cellular pathways in response to environmental and temporal stimuli (1). The control of diverse biochemical pathways, including cell cycle progression and the response to DNA damage, is mediated, at least in part, by dynamic alterations in protein degradation (2). Previous large scale proteomics studies in mammalian cells have shown that the rate of protein degradation can vary from the timescale of minutes, to essentially infinite stability for metastable proteins (3–8).Most intracellular proteins have similar degradation rates, with a half-life approximating the cell doubling rate. Under 5% of proteins display degradation rates more than threefold faster than the proteome average (3–5, 7). However, degradation rates for individual proteins can change, for example depending on either the cell cycle stage, or signaling events, and can also vary depending on subcellular localization. Disruption of such regulated protein stability underlies the disease mechanisms responsible for forms of cancer, e.g. p53 (9, 10) and the proto-oncogene c-Myc (11).Detection of rapidly degraded proteins can be difficult because of their low abundance. However, advances in mass spectrometry based proteomics have enabled in-depth quantitative analysis of cellular proteomes (12–14). Stable isotope labeling by amino acids in cell culture (SILAC)¹ (15), has been widely used to measure protein properties such as abundance, interactions, modifications, turnover, and subcellular localization under different conditions (16). Subcellular fractionation and protein size separation are also powerful techniques that enhance in-depth analysis of cellular proteomes. Not only do these fractionation techniques increase total proteome coverage, they also provide biological insight regarding how protein behavior differs between subcellular compartments. For example, subcellular fractionation has highlighted differences in the rate of ribosomal protein degradation between the nucleus and cytoplasm, (7, 17). Other studies have also demonstrated the benefit of in-depth subcellular fractionation and created methods for the characterization of how proteomes are localized in organelles (18–20).In this study we have used SILAC-based quantitative mass spectrometry combined with extensive subcellular and protein-level fractionation to identify rapidly degraded proteins in human U2OS cells. We provide a proteome level characterization of a major feedback mechanism involving inhibition of protein translation when the proteasome is inhibited. We also present the Encyclopedia of Proteome Dynamics, a user-friendly online resource providing access to the entire data set.     

Design and synthesis of 1,3-biarylsulfanyl derivatives as new anti-breast cancer agents

A new series of 1,3-biarylsulfanyl derivatives (homodibenzyl core motif) have been designed and synthesized as new estrogen receptor ligands by chopping benzothiophene core of raloxifene to engender seco-raloxifene scaffold. All the synthesized compounds were screened for anti-proliferative, anti-osteoporotic, and anti-implantation activity. Compounds (35, 36) having basic amino anti-estrogenic side chain were exhibiting potential anti-proliferative activity in MCF-7, MDA-MB-231 and ishikawa cell lines. Some of the synthesized compounds having homodibenzyl motif (5, 8, 10) have shown moderate anti-osteoporotic activity.