Identification of cis-localization elements of the maize 10-kDa δ-zein and their use in targeting RNAs to specific cortical endoplasmic reticulum subdomains

The RNAs for the storage proteins of rice ( Oryza sativa ), prolamines and glutelins, which are stored as inclusions in the lumen of the endoplasmic reticulum (ER) and storage vacuoles, respectively, are targeted by specific cis -localization elements to distinct subdomains of the cortical ER. Glutelin RNA has one or more cis -localization elements (zip codes) at the 3' end of the RNA, whereas prolamine has two cis -elements; one located in the 5' end of the coding sequence and a second residing in the 3'-untranslated region (UTR). We had earlier demonstrated that the RNAs for the maize zeins ('prolamine' class) are localized to the spherical protein body ER (PB-ER) in developing maize endosperm. As the PB-ER localization of the 10-kDa δ-zein RNA is maintained in developing rice seeds, we determined the number and proximate location of their cis -localization elements by expressing GFP fusions containing various zein RNA sequences in transgenic rice and analyzing their spatial distribution on the cortical ER by in situ RT-PCR and confocal microscopy. Four putative cis -localization elements were identified; three in the coding sequences and one in the 3'-UTR. Two of these zip codes are required for restricted localization to the PB-ER. Using RNA targeting determinants we show, by mis-targeting the storage protein RNAs from their normal destination on the cortical ER, that the coded proteins are redirected from their normal site of deposition. Targeting of RNA to distinct cortical ER subdomains may be the underlying basis for the variable use of the ER lumen or storage vacuole as the final storage deposition site of storage proteins among flowering plant species.     

Stable nuclear transformation of the Closterium peracerosum-strigosum-littorale complex

Although charophycean algae form a relevant monophyly with embryophytes and hence occupy a fundamental place in the development of Streptophyta, no tools for genetic transformation in these organisms have been developed. Here we present the first stable nuclear transformation system for the unicellular Zygnematales, the Closterium peracerosum-strigosum-littorale complex (C. psl complex), which is one of the most useful organisms for experimental research on charophycean algae. When a vector, pSA106, containing the dominant selectable marker ble (phleomycin-resistant) gene and a reporter cgfp (Chlamydomonas-adapted green fluorescent protein) gene was introduced into cells via particle bombardment, a total of 19 phleomycin-resistant cells were obtained in the presence of a low concentration of phleomycin. Six isogenic strains isolated using conditioned medium showed consecutive cgfp expression and long-term stability for phleomycin resistance. DNA analyses verified single or tandem/redundant integration of ~10 copies of pSA106 into the C. psl complex genome. We also constructed an overexpression vector, pSA1102, and then integrated a CpPI gene encoding minus-specific sex pheromone into pSA1102. Ectopic overexpression of CpPI and the pheromonal function were confirmed when the vector pSA1102_CpPI was introduced into mt(+) cells. The present efficient transformation system for the C. psl complex should provide not only a basis for molecular investigation of Closterium but also an insight into important processes in early development and evolution of Streptophyta.     

Structural insights into differences in drug-binding selectivity between two forms of human alpha1-acid glycoprotein genetic variants, the A and F1*S forms

Human α(1)-acid glycoprotein (hAGP) in serum functions as a carrier of basic drugs. In most individuals, hAGP exists as a mixture of two genetic variants, the F1*S and A variants, which bind drugs with different selectivities. We prepared a mutant of the A variant, C149R, and showed that its drug-binding properties were indistinguishable from those of the wild type. In this study, we determined the crystal structures of this mutant hAGP alone and complexed with disopyramide (DSP), amitriptyline (AMT), and the nonspecific drug chlorpromazine (CPZ). The crystal structures revealed that the drug-binding pocket on the A variant is located within an eight-stranded β-barrel, similar to that found in the F1*S variant and other lipocalin family proteins. However, the binding region of the A variant is narrower than that of the F1*S variant. In the crystal structures of complexes with DSP and AMT, the two aromatic rings of each drug interact with Phe-49 and Phe-112 at the bottom of the binding pocket. Although the structure of CPZ is similar to those of DSP and AMT, its fused aromatic ring system, which is extended in length by the addition of a chlorine atom, appears to dictate an alternative mode of binding, which explains its nonselective binding to the F1*S and A variant hAGPs. Modeling experiments based on the co-crystal structures suggest that, in complexes of DSP, AMT, or CPZ with the F1*S variant, Phe-114 sterically hinders interactions with DSP and AMT, but not CPZ.     

Coenzyme Q10 as a potent compound that inhibits Cdt1–geminin interaction

A human replication initiation protein Cdt1 is a very central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by directly binding to it. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1–geminin imbalance, for example by geminin silencing with siRNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. In the present study, we first established a high throughput screening system based on modified ELISA (enzyme linked immunosorbent assay) to identify compounds that interfere with human Cdt1–geminin binding. Using this system, we found that coenzyme Q₁₀ (CoQ₁₀) can inhibit Cdt1–geminin interaction in vitro. CoQ compound is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ₁₀, having a longer isoprenoid chain, was the strongest inhibitor of Cdt1–geminin binding in the tested CoQs, with 50% inhibition observed at concentrations of 16.2 μM. Surface plasmon resonance analysis demonstrated that CoQ₁₀ bound selectively to Cdt1, but did not interact with geminin. Moreover, CoQ₁₀ had no influence on the interaction between Cdt1 and mini-chromosome maintenance (MCM)4/6/7 complexes. These results suggested that CoQ₁₀ inhibits Cdt1–geminin complex formation by binding to Cdt1 and thereby could liberate Cdt1 from inhibition by geminin. Using three-dimensional computer modeling analysis, CoQ₁₀ was considered to interact with the geminin interaction interface on Cdt1, and was assumed to make hydrogen bonds with the residue of Arg243 of Cdt1. CoQ₁₀ could prevent the growth of human cancer cells, although only at high concentrations, and it remains unclear whether such an inhibitory effect is associated with the interference with Cdt1–geminin binding. The application of inhibitors for the formation of Cdt1–geminin complex is discussed.     

Involvement of Protein Kinase C Activation in L-Leucine-Induced Stimulation of Protein Synthesis in L6 Myotubes

Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [³H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, α-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A₂ and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C.     

Identification of 4-Trimethylaminobutyraldehyde Dehydrogenase (TMABA-DH) as a Candidate Serum Autoantibody Target for Kawasaki Disease

Kawasaki disease (KD), an acute vasculitis that preferentially affects coronary arteries, is still the leading cause of acquired heart disease in children. Although the involvement of immune system malfunction in the onset of KD is suggested, its etiology still remains to be clarified. We investigated autoantibodies in KD patients, which are frequently found in sera from patients with autoimmune diseases, vasculitides and arteritides. We performed two-dimensional western blotting and LC-MS/MS to analyze the antigens of autoantibodies, detected two protein spots with 4 out of 24 sera from KD patients but not with 6 control sera, and identified the antigens as 4-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH). A slot blot analysis with TMABA-DH as an antigen also revealed higher reactivities of patients'' sera than control sera (positive rates: 18/43 vs 3/41). Using an enzyme-linked immunosorbent assay (ELISA), we found that the reactivity of anti-TMABA-DH antibodies in sera from KD patients was significantly higher than that in sera from age-matched controls. The optimal cut-off value of 0.043 had a sensitivity of 83.7% and a specificity of 80.0% in detecting KD patients (positive rates: 37/43 for KD patients, 9/41 for controls). Immunohistochemistry performed on thin sections of rat heart revealed that TMABA-DH colocalized with myosin light chains in cardiac myocytes. Patient sera with high reactivity gave similar immunostaining pattern. These results suggest that the detection of anti-TMABA-DH autoantibody could be a potential strategy for a diagnosis of KD.     

Purification and properties of a lytic enzyme from the cell wall of Chlorella ellipsoidea C-87

Cell wall lytic activity was detected in the culture medium and cell wall of 1AM Chlorella ellipsoidea C-87. The enzymes of both fractions had their highest activity at pH 5. The lytic activity bound to the cell wall consisted of a polysaccharide releasing enzyme, an exo-type enzyme releasing disaccharide, and glucosidase; but only the polysaccharide releasing enzyme was solubilized by lithium chloride. A polysaccharide releasing enzyme with a molecular weight around 40 kDa was isolated from the culture medium. Hemicellulose is degraded by the polysaccharide releasing enzyme, and the rigid wall by the exo-type enzyme.