Class VI myosin moves processively along actin filaments backward with large steps.

Among a superfamily of myosin, class VI myosin moves actin filaments backwards. Here we show that myosin VI moves processively on actin filaments backwards with large ( approximately 36 nm) steps, nevertheless it has an extremely short neck domain. Myosin V also moves processively with large ( approximately 36 nm) steps and it is believed that myosin V strides along the actin helical repeat with its elongated neck domain that is critical for its processive movement with large steps. Myosin VI having a short neck cannot take this scenario. We found by electron microscopy that myosin VI cooperatively binds to an actin filament at approximately 36 nm intervals in the presence of ATP, raising a hypothesis that the binding of myosin VI evokes "hot spots" on actin filaments that attract myosin heads. Myosin VI may step on these "hot spots" on actin filaments in every helical pitch, thus producing processive movement with 36 nm steps.     

Diabetic modifier QTLs in F2 intercrosses carrying homozygous transgene of TGF-β

When the homozygous active form of porcine TGF-β1 transgene (Tgf/Tgf) (under control of the rat glucagon promoter) is introduced into the nonobese diabetic mouse (NOD) genetic background, the mice develop endocrine and exocrine pancreatic hypoplasia, low serum insulin concentrations, and impaired glucose tolerance. To identify genetic modifiers of the diabetic phenotypes, we crossed hemizygous NOD-Tgf with DBA/2J mice (D2) or C3H/HeJ mice (C3H) and used the “transgenic mice” for quantitative trait loci (QTL) analysis. Genome-wide scans of F₂-D Tgf/Tgf (D2 × NOD) and F₂-C Tgf/Tgf (C3H × NOD), homozygous for the TGF-β1 transgene, identified six statistically significant modifier QTLs: one QTL (Tdn1) in F₂-D Tgf/Tgf, and five QTLs (Tcn1 to Tcn5) in F₂-C Tgf/Tgf. Tdn1 (Chr 13, LOD = 4.39), and Tcn3 (Chr 2, LOD = 4.94) showed linkage to body weight at 8 weeks of age. Tcn2 (Chr 7, LOD = 4.38) and Tcn4 (Chr 14, LOD = 3.99 and 3.78) showed linkage to blood glucose (BG) concentrations in ipGTT at 30, 0, and 120 min, respectively. Tcn1 (Chr 1, LOD = 4.41) and Tcn5 (Chr 18, LOD = 4.99) showed linkage to serum insulin concentrations in ipGTT at 30 min. Tcn2 includes the candidate gene, uncoupling protein 2 (Ucp2), and shows linkage to Ucp2 mRNA levels in the soleus muscle (LOD = 4.90). Identification of six QTLs for diabetes-related traits in F₂-D Tgf/Tgf and F₂-C Tgf/Tgf raises the possibility of identifying candidate susceptibility genes and new targets for drug development for human type 2 diabetes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Design, synthesis, and biological evaluation of 4-phenylpyrrole derivatives as novel androgen receptor antagonists

A series of 4-phenylpyrrole derivatives D were designed, synthesized, and evaluated for their potential as novel orally available androgen receptor antagonists therapeutically effective against castration-resistant prostate cancers. 4-Phenylpyrrole compound 1 exhibited androgen receptor (AR) antagonistic activity against T877A and W741C mutant-type ARs as well as wild-type AR. An arylmethyl group incorporated into compound 1 contributed to enhancement of antagonistic activity. Compound 4n, 1-{[6-chloro-5-(hydroxymethyl)pyridin-3-yl]methyl}-4-(4-cyanophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonitrile exhibited inhibitory effects on tumor cell growth against the bicalutamide-resistant LNCaP-cxD2 cell line as well as the androgen receptor-dependent JDCaP cell line in a mouse xenograft model. These results demonstrate that this series of pyrrole compounds are novel androgen receptor antagonists with efficacy against prostate cancer cells, including castration-resistant prostate cancers such as bicalutamide-resistant prostate cancer.     

Long chain fatty acid synthesis in developing castor bean seeds I. The operation of the path from acetate to long chain fatty acids in a subcellular particulate system

The path of LFA synthesis from acetate in developing castorbean seeds was associated with subcellular 10,000 g particles.Further fractionation of these particles by a stepwise densitygradient method showed the high possibility that the site ofLFA synthesis is the proplastid. A study on cofactor requirementswhen [1-¹⁴C]acetate predominantly incorporated into LFAs indicatedthat synthesis would be achieved by acetyl-CoA carboxylation,malonyl-ACP condensation. ATP, CoA, HCO₃^– and Mg⁺⁺ orMn⁺⁺ were essential for synthesis from acetate by the 10,000gparticulate system. Results of inhibhitor experiment suggestedthat the supply of ATP to the LFA synthesizing system is broughtabout by mitochondrial oxidative phosphorylation, when acetateis the sole precursor for LFA synthesis in this system. TheNADPH generating system was contained in the paticles, althoughthe addition of NADP⁺ and G-6-P increased synthesis. NADH markedlystimulated LFA synthesis from acetate. The primary role of NADHseems to be as a direct reductant in both steps involving thereduction and oxidative desaturation of fatty acid chains; particularly,in the former step, although NADH partially contributes to thesupply of ATP as a respiratory substrate. It is unlikely thatNADH works as a hydrogen donor to NADP⁺. LFA synthesis by thecastor bean particulate system was not stimulated by light,thus differing from that by leaf chloroplasts. (Received July 23, 1973; )     

Proteasome-dependent degradation of alpha-catenin is regulated by interaction with ARMc8alpha

ARMc8 (armadillo-repeat-containing protein 8) is a key component of the CTLH (C-terminal to lissencephaly type-1-like homology motif) complex in mammalian cells. This complex is well conserved in Saccharomyces cerevisiae and has been characterized as a FBPase (fructose-1, 6-bisphosphatase)-degrading complex. The yeast homologue of ARMc8, Gid (glucose-induced degradation) 5p, plays an essential role in the ubiquitin- and proteasome-dependent degradation of FBPase. To elucidate the function of ARMc8, we used a yeast two-hybrid system to screen a human skeletal muscle cDNA library. alpha-Catenin was isolated as a binding protein of ARMc8alpha. This association was confirmed by co-immunoprecipitation assay using MDCK (Madin-Darby canine kidney) cells in which exogenous alpha-catenin and ARMc8alpha were overexpressed. The association was also confirmed by co-immunoprecipitation assay using endogenous proteins in untransfected MDCK cells. We then used immunofluorescence microscopy of MDCK cells and C2C12 cells to investigate the intracellular distribution of ARMc8. Exogenously expressed ARMc8 was co-localized with alpha-catenin and beta-catenin along the cell membrane, suggesting an association between alpha-catenin and ARMc8 in the cells. To compare the binding domain of alpha-catenin with ARMc8alpha with that of beta-catenin, we performed a co-immunoprecipitation assay, again using 5'- and 3'-deletion constructs of alpha-catenin. The N-terminal sequence (amino acids 82-148) of alpha-catenin was sufficient to bind to both ARMc8alpha and beta-catenin. Next, we investigated the proteasome-dependent degradation of alpha-catenin by immunoblotting using proteasome inhibitors. Co-expression of ARMc8alpha with alpha-catenin resulted in rapid degradation of the exogenous alpha-catenin. Furthermore, ARMc8 knockdown inhibited alpha-catenin degradation and prolonged the half-life of alpha-catenin. We conclude that ARMc8alpha associates with alpha-catenin and up-regulates its degradation.     

Dual roles of photosynthetic electron transport in photosystem I biogenesis: light induction of mRNAs and chromatic regulation at post-mRNA level

Light regulation of photosystem I (PSI) biogenesis was studied in a unicellular green alga, Chlamydomonas reinhardtii. When Chlamydomonas cells were transferred from darkness to the light, mRNAs for both nuclear- and chloroplast-encoded PSI subunits were induced in concert. This light induction was inhibited by photosynthetic electron transport (PET) inhibitors, 3-(3,4 dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6 isopropyl-p-benzoquinone, but not by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone. This indicated that PET plays a pivotal role in the light induction of PSI subunit mRNAs, but that photophosphorylation is not necessary. When we irradiated the Chlamydomonas cells with PSI-light (695 nm) or PSII-light (644 nm), which makes the plastoquinone pool oxidative and reductive, respectively, PSII-light caused the accumulation of PSI proteins more abundantly than did PSI-light. However, there was no difference for the PSI subunit mRNA levels between these light sources. From these results, we conclude that PET plays dual roles in the regulation of PSI biogenesis in Chlamydomonas: when cells are illuminated, PET first induces the PSI subunit mRNAs irrespective of the redox state of the intersystem electron carriers, and then their redox state fine-tunes PSI biogenesis at translational and/or post-translational steps to fulfil the chromatic adaptation.