Small molecule Y-320 stimulates ribosome biogenesis,protein synthesis,and aminoglycoside-induced premature termination codon readthrough

Premature termination codons (PTC) cause over 10% of genetic disease cases. Some aminoglycosides that bind to the ribosome decoding center can induce PTC readthrough and restore low levels of full-length functional proteins. However, concomitant inhibition of protein synthesis limits the extent of PTC readthrough that can be achieved by aminoglycosides like G418. Using a cell-based screen, we identified a small molecule, the phenylpyrazoleanilide Y-320, that potently enhances TP53, DMD, and COL17A1 PTC readthrough by G418. Unexpectedly, Y-320 increased cellular protein levels and protein synthesis, measured by SYPRO Ruby protein staining and puromycin labeling, as well as ribosome biogenesis measured using antibodies to rRNA and ribosomal protein S6. Y-320 did not increase the rate of translation elongation and it exerted its effects independently of mTOR signaling. At the single cell level, exposure to Y-320 and G418 increased ribosome content and protein synthesis which correlated strongly with PTC readthrough. As a single agent, Y-320 did not affect translation fidelity measured using a luciferase reporter gene but it enhanced misincorporation by G418. RNA-seq data showed that Y-320 up-regulated the expression of CXC chemokines CXCL10, CXCL8, CXCL2, CXCL11, CXCL3, CXCL1, and CXCL16. Several of these chemokines exert their cellular effects through the receptor CXCR2 and the CXCR2 antagonist SB225002 reduced cellular protein levels and PTC readthrough in cells exposed to Y-320 and G418. These data show that the self-limiting nature of PTC readthrough by G418 can be compensated by Y-320, a potent enhancer of PTC readthrough that increases ribosome biogenesis and protein synthesis. They also support a model whereby increased PTC readthrough is enabled by increased protein synthesis mediated by an autocrine chemokine signaling pathway. The findings also raise the possibility that inflammatory processes affect cellular propensity to readthrough agents and that immunomodulatory drugs like Y-320 might find application in PTC readthrough therapy.

A cell-based screen identifies a small molecule, Y-320, that potently enhances readthrough of premature termination codons by the aminoglycoside G418. Y-320 increases cellular protein levels and protein synthesis, and appears to act via an autocrine chemokine signaling pathway.     

Immunodetection of low concentrations of ovalbumin in cryoprocessed quail oviduct cells by semi-automatic quantitative analysis

Summary— In a previous paper (Quintana et al, Biol Cell 72, (1991) 167–180) we reported the anti-ovalbumin antibody immunolabeling in the cytoplasm of tubular gland cells from cryofixed, cryosubstituted and acrylic-resin embedded quail oviduct. To confirm these preliminary visual observations, we have carried out a semi-automatic quantitative study of immunogold labeling. The quantitative results confirm the previous data. In addition, we found a significantly higher immunolabeling with low temperature Lowicryl K11M embedding procedure as compared with high temperature LR White embedding.     

A method of comparing spermatozoa with light and scanning electron microscopy

A new method of comparing light microscopy and scanning electron microscopy in the study of small cells, such as spermatozoa, that must be examined under oil immersion is described. A grid is etched on the corner of a microscope glass slide, and its inner edges are incised. Its surface area is calculated as a function f the chamber of the critical-point drying apparatus. This method dispenses with the need for any special coverslip and enables the cells to be observed under oil immersion.     

Formation of polarized contractile interfaces by self-organized Toll-8/Cirl GPCR asymmetry

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Simple preparation of 1,2-dipalmitoyl-sn-glycero-3-phosphoric acid and deuterated choline derivatives

Analytically pure 1,2-dipalmitoyl-sn-glycero-3-phosphoric acid was prepared in gram amounts, using a simplified version of a previous procedure. The main step, enzymatic cleavage of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with freshly extracted phospholipase D, was performed in the presence of chloroform and the crude phosphatidic acid was purified by silica gel column chromatography. The interest of the method was illustrated by the synthesis of two dipalmitoyl phosphatidylcholines selectively deuterated on the polar headgroup.     

Mechano-induced cell metabolism promotes microtubule glutamylation to force metastasis

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Regio- and stereo-selectivity in the induction of peroxisome proliferation by substituted hexanoic acids

Summary— Quantitative structure-activity relationship is an effective tool in order to predict drug potency. A similar approach is actually developed for peroxisome proliferation induced by substituted carboxylic acids issued from plasticizer metabolism in rats. The study is focused on acids found in rat urine after adipic diester dosings. Size, location of the substituted group and length of the chain have been studied. 3-D structure has also been taken in account for 2-ethyl hexanoic acids. The results obtained so far demonstrate that peroxisome proliferation potencies of the considered acids are modified according structure changes. At this time location of the group along the chain appears to be a predominant factor.     

Differential susceptibility to biological detergents of the particulate cGMP-stimulated phosphodiesterase from rat heart: preservation of the allosteric properties of the solubilized enzyme

The effects of various biological detergents on the particulate cGMP-stimulated cAMP phosphodiesterase activity from rat heart were investigated. When added to particulate fractions, anionic and non-ionic detergents diversely increased both cAMP and cGMP phosphodiesterase activities and slightly decreased the stimulatory effect of cGMP on cAMP hydrolysis whereas cationic detergents were rather inhibitory and drastically lowered the stimulatory effect of cGMP. Among the most efficient detergents, only sodium cholate was able to solubilize phosphodiesterase activity and preserve the stimulatory effect of cGMP on cAMP hydrolysis. Furthermore, the addition of glycerol significantly improved the conservation of the allosteric properties of the enzyme. Kinetic properties of the cholate-solubilized phosphodiesterase were quite identical to those of the membrane-bound enzyme.     

Evidence for Two Main Domestication Trajectories in Saccharomyces cerevisiae Linked to Distinct Bread-Making Processes

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