Catalyst-Directed Guidance of Sulfur-Substituted Enediolates to Stereoselective Carbon–Carbon Bond Formation with Aldehydes

A highly chemo-, regio-, and stereoselective glycolate aldol reaction of sulfur-substituted enediolates with aldehydes was developed by employing a l-cyclohexylglycine-derived chiral iminophosphorane as a catalyst. The key for establishing this protocol is the distinct ability of the iminophosphorane catalyst to precisely direct the equilibrium mixture of the enediolates toward the intermolecular carbon–carbon bond formation with simultaneous yet rigorous control of relative and absolute stereochemistry. The critical importance of the cyclohexyl substituents on the catalyst backbone in dictating the reaction pathway and the stereochemical outcome was elucidated through an extensive quantum analysis by density functional theory calculations

Catalyst-Directed Guidance of Sulfur-Substituted Enediolates to Stereoselective Carbon–Carbon Bond Formation with Aldehydes

A highly chemo-, regio-, and stereoselective glycolate aldol reaction of sulfur-substituted enediolates with aldehydes was developed by employing a l-cyclohexylglycine-derived chiral iminophosphorane as a catalyst. The key for establishing this protocol is the distinct ability of the iminophosphorane catalyst to precisely direct the equilibrium mixture of the enediolates toward the intermolecular carbon–carbon bond formation with simultaneous yet rigorous control of relative and absolute stereochemistry. The critical importance of the cyclohexyl substituents on the catalyst backbone in dictating the reaction pathway and the stereochemical outcome was elucidated through an extensive quantum analysis by density functional theory calculations

Catalyst-Directed Guidance of Sulfur-Substituted Enediolates to Stereoselective Carbon–Carbon Bond Formation with Aldehydes

A highly chemo-, regio-, and stereoselective glycolate aldol reaction of sulfur-substituted enediolates with aldehydes was developed by employing a l-cyclohexylglycine-derived chiral iminophosphorane as a catalyst. The key for establishing this protocol is the distinct ability of the iminophosphorane catalyst to precisely direct the equilibrium mixture of the enediolates toward the intermolecular carbon–carbon bond formation with simultaneous yet rigorous control of relative and absolute stereochemistry. The critical importance of the cyclohexyl substituents on the catalyst backbone in dictating the reaction pathway and the stereochemical outcome was elucidated through an extensive quantum analysis by density functional theory calculations

Catalyst-Directed Guidance of Sulfur-Substituted Enediolates to Stereoselective Carbon–Carbon Bond Formation with Aldehydes

A highly chemo-, regio-, and stereoselective glycolate aldol reaction of sulfur-substituted enediolates with aldehydes was developed by employing a l-cyclohexylglycine-derived chiral iminophosphorane as a catalyst. The key for establishing this protocol is the distinct ability of the iminophosphorane catalyst to precisely direct the equilibrium mixture of the enediolates toward the intermolecular carbon–carbon bond formation with simultaneous yet rigorous control of relative and absolute stereochemistry. The critical importance of the cyclohexyl substituents on the catalyst backbone in dictating the reaction pathway and the stereochemical outcome was elucidated through an extensive quantum analysis by density functional theory calculations

Catalyst-Directed Guidance of Sulfur-Substituted Enediolates to Stereoselective Carbon–Carbon Bond Formation with Aldehydes

A highly chemo-, regio-, and stereoselective glycolate aldol reaction of sulfur-substituted enediolates with aldehydes was developed by employing a l-cyclohexylglycine-derived chiral iminophosphorane as a catalyst. The key for establishing this protocol is the distinct ability of the iminophosphorane catalyst to precisely direct the equilibrium mixture of the enediolates toward the intermolecular carbon–carbon bond formation with simultaneous yet rigorous control of relative and absolute stereochemistry. The critical importance of the cyclohexyl substituents on the catalyst backbone in dictating the reaction pathway and the stereochemical outcome was elucidated through an extensive quantum analysis by density functional theory calculations

<i>Tol2</i> knockdown vectors were genomically integrated and the inducible expression was tightly controlled <i>in vivo</i>.

<p>(A) Representative neocortical sections showing the basal expression and induced expression of EGFP derived from the pT2K-BI-shRNAmir or pT2K-TBI-shRNAmir vectors. (B) The retention and expression of inducible knockdown vector in glial cells. In the presence of <i>Tol2</i> transposase, EGFP was observed in the glial cells (arrowheads). The right panels are higher magnification views of the boxed regions in the left panels. (C) Representative neocortical sections showing the retention and expression of the inducible knockdown vector in the adult cortex. CP, cortical plate; VZ, ventricular zone. Scale bars, 100 µm.</p

The <i>Tol2</i> transposable vector enables inducible knockdown from a stably integrated knockdown cassette.

<p>(A) A schematic diagram of the pT2K-TBI-shRNAmir vector. The shRNAmir cassette was inserted into the pT2K-BI-TRE-EGFP vector, and TRE-BI was replaced with TRE-TBI. The shRNAmir cassette consisted of the hairpin stem, which is composed of siRNA sense and antisense strands designed for the knockdown of the target gene, a loop derived from human mir30, and mir30 flanking sequences on the 3′ and 5′ sides of the hairpin. (B) A schematic diagram showing the principle of induction of knockdown from the genomically integrated shRNAmir cassette. The <i>Tol2</i>-flanked region of the plasmids were excised and integrated into the chromosome using <i>Tol2</i> transposase. In the presence of Doxycycline (Dox), rtTA-M2 bound to TRE-TBI, and the expression of both EGFP and the mir30-based knockdown cassette were induced under the control of TRE-TBI. (C) Expression of EGFP, induced from the each of pT2K-TBI-shRNAmir vectors (mir-empty, mir-APP#2 and mir-APP#3), was observed in almost all PC12 cells following Dox administration. The upper panels show the bright-field images. Scale bar, 100 µm. (D) Immunoblot analyses for evaluating the knockdown efficiency against APP. Actin was used as a loading control. (E) The basal expression (−Dox) and the induced expression (+Dox) of EGFP from the pT2K-BI-shRNAmir and pT2K-TBI-shRNAmir vectors in HEK293T cells. pCAGGS-tdTomato was co-transfected as a transfection control. Inset shows a higher magnification. Scale bars: 100 µm, inset 20 µm. (F) Ratio of the number of EGFP-positive cells to tdTomato-positive cells between the cells expressing pT2K-BI-shRNAmir and pT2K-TBI-shRNAmir with or without Dox. (mean ± SEM, n = 3). Abbreviations of the vector name and their components are listed in the table (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033380#pone.0033380.s002" target="_blank">Table S1</a>).</p

The SMC_prok_B domain of FILIP is responsible for the modification of actin stress fibers.

<p>(A) We observed no apparent actin stress fibers in FILIP-expressing NIH3T3 cells. In contrast, actin stress fibers were clearly visible in FILIP d248-685-expressing NIH3T3 cells. Actin fibers were visualized (red) using Alexa 568-conjugated phalloidin. Green, bicistronic GFP expression vector of FILIP. Scale bar = 10 μm. (B) The graph shows the ratio of cells containing actin stress fibers to total cells examined. The ratios were 345/409 for control cells, 233/413 for FILIP-expressing cells, 405/487 for FILIP d248-685-expressing cells, and 208/350 for FILIP d872-1111-expressing cells. *p < 0.01 (Fisher’s exact test).</p

Subcellular distribution of non-muscle myosin IIb is controlled by FILIP through Hsc70

<div><p>The neuronal spine is a small, actin-rich dendritic or somatic protrusion that serves as the postsynaptic compartment of the excitatory synapse. The morphology of the spine reflects the activity of the synapse and is regulated by the dynamics of the actin cytoskeleton inside, which is controlled by actin binding proteins such as non-muscle myosin. Previously, we demonstrated that the subcellular localization and function of myosin IIb are regulated by its binding partner, filamin-A interacting protein (FILIP). However, how the subcellular distribution of myosin IIb is controlled by FILIP is not yet known. The objective of this study was to identify potential binding partners of FILIP that contribute to its regulation of non-muscle myosin IIb. Pull-down assays detected a 70-kDa protein that was identified by mass spectrometry to be the chaperone protein Hsc70. The binding of Hsc70 to FILIP was controlled by the adenosine triphosphatase (ATPase) activity of Hsc70. Further, FILIP bound to Hsc70 via a domain that was not required for binding non-muscle myosin IIb. Inhibition of ATPase activity of Hsc70 impaired the effect of FILIP on the subcellular distribution of non-muscle myosin IIb. Further, in primary cultured neurons, an inhibitor of Hsc70 impeded the morphological change in spines induced by FILIP. Collectively, these results demonstrate that Hsc70 interacts with FILIP to mediate its effects on non-muscle myosin IIb and to regulate spine morphology.</p></div

Inhibition of Hsc70 results in the suppression of the effects of FILIP on the subcellular distribution of non-muscle myosin IIb.

<p>A-D: The graphs show the ratio of the COS-7 cells exhibiting a stress fiber-like distribution versus a granular distribution of non-muscle myosin IIb (A) after treatment with 2 mM clofibric acid (numbers of cells containing a stress fiber-like distribution/total cells: 543/625 control cells treated with vehicle; 571/615 control cells treated with clofibric acid; 182/628 FILIP-expressing cells treated with vehicle; and 243/620 FILIP-expressing cells treated with clofibric acid. *p < 0.01 (Fisher’s exact test)); (B) under the expression of FILIP d687-960 with or without the application of clofibric acid (numbers of stress fiber-like distributed cells/total cells: 166/318 FILIP d687-960-expressing cells treated with vehicle and 158/307 FILIP d687-960-expressing cells treated with clofibric acid); (C) under the expression of FILIP d872-1111 with or without the application of clofibric acid (numbers of stress fiber-like distributed cells/total cells: 178/325 FILIP d872-1111-expressing cells treated with vehicle and 185/331 FILIP d872-1111-expressing cells treated with clofibric acid); and (D) under the expression of Hsc70K71M and FILIP (numbers of non-muscle myosin IIb stress fiber-like distributed cells 273/320 control/Hsc70-expressing cells; 282/320 control/Hsc70K71M-expressing cells, 131/340 FILIP/Hsc70-expressing cells, and 151/318 FILIP/Hsc70K71M-expressing cells. (E) Mutation of Hsc70 does not influence the binding of FILIP and non-muscle myosin IIb. Immunoprecipitation was performed using an anti-FLAG antibody, and blots were probed with an antibody against non-muscle myosin IIb.</p