Regulation of Retention of FosB Intron 4 by PTB

One effect of stressors such as chronic drug administration is that sequence within the terminal exon of the transcription factor FosB is recognized as intronic and removed by alternative splicing. This results in an open-reading-frame shift that produces a translation stop codon and ultimately a truncated protein, termed ΔFosB. In vitro splicing assays with control and mutated transcripts generated from a fosB mini-gene construct indicated a CU-rich sequence at the 3′ end of intron 4 (I4) plays an important role in regulating fosB pre-mRNA splicing due to its binding of polypyrimidine tract binding protein (PTB). PTB binding to this sequence is dependent upon phosphorylation by protein kinase A and is blocked if the CU-rich sequence is mutated to a U-rich region. When this mutated fosB minigene is expressed in HeLa cells, the splicing efficiency of its product is increased compared to wild type. Moreover, transient transfection of PTB-1 in HeLa cells decreased the splicing efficiency of a wild type fosB minigene transcript. Depletion of PTB from nuclear extracts facilitated U2AF65 binding to wild type sequence in vitro, suggesting these proteins function in a dynamic equilibrium to modulate fosB pre-mRNA alternative splicing. These results demonstrate for the first time that phosphorylated PTB promotes intron retention and thereby silences the splicing of fosB I4

MODELING AND ANALYSIS OF KNEE - DASHBOARD IMPACT IN FRONTAL COLLISION

During the frontal collision of a vehicle, be it with another vehicle or with a fixed or mobile obstacle, one of the most frequent injuries suffered by the driver of the vehicle occurs in the knee joint. Thus, the present paper aims at investigating the knee to dashboard contact during a frontal collision. Admittedly, the first stage of our investigation underpinned the design of a model to encompass the dummy and the main interior elements of the vehicle. Subsequently, the main types of contacts provided by the LS-DYNA software have been analysed. In order to establish the optimal modelling variant of the impact between the knee and the dashboard various simulations have been carried out while applying different methods to define the contact. The results obtained following the simulations have been then compared to the results provided by the specialized literature

Syntheses of Variations of Stereogenic-at-Metal Imido Alkylidene Complexes of Molybdenum

In this paper we describe the syntheses of several new stereogenic-at-metal imido alkylidene complexes of molybdenum, Mo(NR)(CHR′)(X)(Y), many of which had to be prepared through selective nucleophilic displacement reactions in imido alkylidene complexes. The reported compounds include Mo(NAd)(CHCMe[subscript 2]Ph)(MesPyr)[subscript 2] (1a; MesPyr = 2-mesitylpyrrolide, Ad = 1-adamantyl), Mo(NAd)(CHCMe[subscript 2]Ph)(2-CNPyr)[subscript 2] (1b; 2-CNPyr = 2-cyanopyrrolide), Mo(NAd)(CHCMe[subscript 2]Ph)(MesPyr)(OTPP) (2a; OTPP = 2,3,5,6-tetraphenylphenoxide), Mo(NAd)(CHCMe[subscript 2]Ph)(MesPyr)(OBr[subscript 2]Bitet) (2b; OBr[subscript 2]Bitet = (R)-3,3′-dibromo-2′-(tert-butyldimethylsilyloxy)-5,5′,6,6′,7,7′,8,8′-octahydro-1,1′-binaphthyl-2-olate), Mo(NAd)(CHCMe[subscript 2]Ph)(OHIPT)(2-Mespyr) (2c; HIPT = 2,6-(2,4,6-iPr[subscript 3]C[subscript 6]H[subscript 2])[subscript 2]C[subscript 6]H[subscript 3]), Mo(NAd)(CHCMe[subscript 2]Ph)(OTf)(OHIPT) (3), Mo(NAd)(CHCMe[subscript 2]Ph)(OTf)(OHIPT)(PMe[subscript 3]) (3(PMe[subscript 3])), Mo(NAd)(CHCMe[subscript 2]Ph)(2-CNPyr)(OHIPT) (4), Mo(NAd)(CHCMe[subscript 2]Ph)(OHIPT)(OCMe[subscript 3]) (5), Mo(NR)(CHCMe[subscript 2]Ph)(OR[subscript F6])(OHMT) (OR[subscript F6] = OCMe(CF[subscript 3])[subscript 2]; HMT = 2,6-Mes[subscript 2]C[subscript 6]H[subscript 3]; R = 2,6-iPr[subscript 2]C[subscript 6]H[subscript 3] (Ar, 6a), 2,6-Me[subscript 2]C[subscript 6]H[subscript 3] (Ar′, 6b), 2-iPrC[subscript 6]H[subscript 4] (Ar[sueprscript iPr], 6c), Ad (6d)), Mo(NR)(CHCMe[subscript 2]Ph)(OR[subscript F6])[N(H)HMT] (7a (R = Ar′) and 7b (R = Ar[superscript iPr])), and Mo(NAd)(CHCMe[subscript 2]Ph)(OR[subscript F6])(HMT) (8). X-ray structural studies were carried out on 1b, 2a–c, 3(PMe[subscript 3]), 4, 5, 6d, 7b, and 8. Compound 1b is an octamer in which two η[superscript 1]-pyrrolides are trans to one another at each metal center and cyano groups bind from neighboring Mo centers trans to the alkylidene and imido ligands.National Science Foundation (U.S.) (CHE-0841187)National Science Foundation (U.S.) (CHE-0946721)National Science Foundation (U.S.) (CHE-111133)National Science Foundation (U.S.) (CHE-9808061

Syntheses of Variations of Stereogenic-at-Metal Imido Alkylidene Complexes of Molybdenum

In this paper we describe the syntheses of several new stereogenic-at-metal imido alkylidene complexes of molybdenum, Mo(NR)(CHR′)(X)(Y), many of which had to be prepared through selective nucleophilic displacement reactions in imido alkylidene complexes. The reported compounds include Mo(NAd)(CHCMe[subscript 2]Ph)(MesPyr)[subscript 2] (1a; MesPyr = 2-mesitylpyrrolide, Ad = 1-adamantyl), Mo(NAd)(CHCMe[subscript 2]Ph)(2-CNPyr)[subscript 2] (1b; 2-CNPyr = 2-cyanopyrrolide), Mo(NAd)(CHCMe[subscript 2]Ph)(MesPyr)(OTPP) (2a; OTPP = 2,3,5,6-tetraphenylphenoxide), Mo(NAd)(CHCMe[subscript 2]Ph)(MesPyr)(OBr[subscript 2]Bitet) (2b; OBr[subscript 2]Bitet = (R)-3,3′-dibromo-2′-(tert-butyldimethylsilyloxy)-5,5′,6,6′,7,7′,8,8′-octahydro-1,1′-binaphthyl-2-olate), Mo(NAd)(CHCMe[subscript 2]Ph)(OHIPT)(2-Mespyr) (2c; HIPT = 2,6-(2,4,6-iPr[subscript 3]C[subscript 6]H[subscript 2])[subscript 2]C[subscript 6]H[subscript 3]), Mo(NAd)(CHCMe[subscript 2]Ph)(OTf)(OHIPT) (3), Mo(NAd)(CHCMe[subscript 2]Ph)(OTf)(OHIPT)(PMe[subscript 3]) (3(PMe[subscript 3])), Mo(NAd)(CHCMe[subscript 2]Ph)(2-CNPyr)(OHIPT) (4), Mo(NAd)(CHCMe[subscript 2]Ph)(OHIPT)(OCMe[subscript 3]) (5), Mo(NR)(CHCMe[subscript 2]Ph)(OR[subscript F6])(OHMT) (OR[subscript F6] = OCMe(CF[subscript 3])[subscript 2]; HMT = 2,6-Mes[subscript 2]C[subscript 6]H[subscript 3]; R = 2,6-iPr[subscript 2]C[subscript 6]H[subscript 3] (Ar, 6a), 2,6-Me[subscript 2]C[subscript 6]H[subscript 3] (Ar′, 6b), 2-iPrC[subscript 6]H[subscript 4] (Ar[sueprscript iPr], 6c), Ad (6d)), Mo(NR)(CHCMe[subscript 2]Ph)(OR[subscript F6])[N(H)HMT] (7a (R = Ar′) and 7b (R = Ar[superscript iPr])), and Mo(NAd)(CHCMe[subscript 2]Ph)(OR[subscript F6])(HMT) (8). X-ray structural studies were carried out on 1b, 2a–c, 3(PMe[subscript 3]), 4, 5, 6d, 7b, and 8. Compound 1b is an octamer in which two η[superscript 1]-pyrrolides are trans to one another at each metal center and cyano groups bind from neighboring Mo centers trans to the alkylidene and imido ligands.National Science Foundation (U.S.) (CHE-0841187)National Science Foundation (U.S.) (CHE-0946721)National Science Foundation (U.S.) (CHE-111133)National Science Foundation (U.S.) (CHE-9808061

PTB and U2AF⁶⁵ bind to the 3′ end of fosB I4.

<p>(A) ³²P-labeled mini RNA substrate transcribed from the 3′ region of I4 within the ISS/WT construct were incubated in the presence of either control HeLa nuclear extract (lanes 1 and 3) or PTB-depleted HeLa nuclear extract (lanes 2 and 4) before UV cross-linking. The samples were then immunoprecipitated with either an α-PTB antibody (lanes 1 and 2) or an α-U2AF⁶⁵ antibody (lanes 3 and 4). Samples were resolved by electrophoresis and visualized by autoradiography. (B) ³²P-labeled mini RNA substrates transcribed from the 3′ region of I4 within either the ISS/WT (lanes 1 and 3) or ISS/T6 constructs (lanes 2 and 4) were incubated in the presence of HeLa nuclear extract before UV cross-linking. The samples were then immunoprecipitated with either an α-PTB antibody (lanes 1 and 2) or an α-U2AF⁶⁵ antibody (lanes 3 and 4), resolved by electrophoresis and visualized by autoradiography.</p

Oligodeoxynucleotides used in subcloning, PCR, UV cross-linking and mutagenesis studies.

<p>Oligodeoxynucleotides used in subcloning, PCR, UV cross-linking and mutagenesis studies.</p

Alkaline phosphatase treatment results in the lack of PTB binding to the 3′ end of fosB I4.

<p>³²P-labeled mini RNA substrates from the 3′ region of fosB I4 were incubated in the presence of purified his-tagged PTB 1 protein and UV cross-linked (lane 1, X). Alternatively, samples were phosphatase treated either after or before the UV cross-linking step (lanes 2 and 3, XP and PX, respectively) or phosphatase digested and kinase treated before or after cross-linking (lanes 4 and 5, PKX and XPK, respectively). Samples were resolved by gel electrophoresis and visualized by autoradiography. The position of a protein molecular weight marker is indicated on the right in kDa.</p