Regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) by Src involves tyrosine phosphorylation of PDK1 and Src homology 2 domain binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr(9) and Tyr(373/376)) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr(9) antibodies showed that the level of Tyr(9) phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr(9), distinct from Tyr(373/376), is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr(9) phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.     

Importance of the degree of antigen polymerization by detoxification in modulating the immunogenicity of acellular pertussis vaccine

For the acellular pertussis vaccine with a high immunogenicity, the concentration, composition and characteristics of acellular pertussis antigens are the crucial points to be considered. Nevertheless, it has not been proved yet whether or not the polymerization degree, one of the characteristics of formalin-detoxified acellular pertussis antigens, has an influence on vaccine potency. Thus, in the present study, the correlations among detoxification conditions of acellular pertussis bulks, their polymerization degrees and their immunogenicities were examined. In addition, the relative importance of pertussis toxoid in vaccine immunogenicity was also investigated. Results show that a lower lysine concentration during detoxification induces highly-polymerized antigens, the immunogenicity has a great dependency on the polymerization degree of antigens, and also pertussis toxoid has a relatively stronger influence on the immunogenicity than other antigens. Accordingly, in the aspect of the potency of detoxified acellular pertussis vaccine, it can be demonstrated that the polymerization of antigens and its degree are the major factors affecting the immunogenicity along with a relatively high content of pertussis toxoid.     

Enzymatic synthesis of amino acid-sugar alcohol conjugates in organic media

Amino acid-sugar alcohol conjugates were synthesized by a commercial serine protease, Optimase M-440, in organic media. Optimase M-440 showed broad substrate specificity towards N-t-Boc-protected l-amino acids as acyl donors and sugar alcohols as nucleophiles. Among various solvents tested Optimase M-440 showed the highest activity in pyridine. The regioselective acylation of the primary –OH groups of sugar alcohols gave the amino acid conjugates in good yields without byproducts.     

Concentration of 6-aminopenicillanic acid from penicillin bioconversion solution and its mother liquor by nanofiltration membrane

In this study, nanofiltration was applied to the concentration of the 6-aminopenicillinic acid (6-APA) from bioconverted penicillin solution and also to its mother liquor. The 6-APA in the solution was concentrated from 0.211 mol/L to 0.746 mol/L by nanofiltration. The final maximum concentration was 3.6 times higher than the initial concentration and the recovery yield was 97% to 99% of the original 6-APA. The concentrated solution was crystallized with the yields of 88.9–90.2% and the purity of the crystallized product was about 98%. The concentration of 6-APA in the mother liquor after crystallization was 0.014 mol/L and thus was concentrated 20–30 fold by nanofiltration and crystallization. The recovery of 6-APA was over 98%. The salts contained in the mother liquor, such as NH₄Cl and KCl, could be removed by allowing them to permeate through the membrane.     

Novel Interaction of the Z-DNA Binding Domain of Human ADAR1 with the Oncogenic c-Myc Promoter G-Quadruplex

Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZα_ADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII₁ region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZα_ADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZα_ADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZα_ADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZα_ADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZα_ADAR1 suppresses the c-myc expression promoted by NHEIII₁ region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.     

Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases

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Effects of Atmospheric O3 on Azolla-Anabaena Symbiosis

Cultures of water fern Azolla pinnata R. Br. exposed for 1 weekto either 30, 50 or 80 nl l^-1 O₃ showed significant reductionsin rates of growth and N₂ fixation, and had fewer heterocysts.Although the levels of glutamine synthetase (GS) and glutamatedehydrogenase (GDH) activity were decreased by low concentrationsof O₃ exposures (30 or 50 nl l^-1), significant increases inlevels of the same enzymes were caused by higher concentrationsof O₃ (80 nl l^-1). Increased levels of total protein, polyamines(putrescine and spermidine), and the xanthophyll-cycle precursorof abscisic acid (ABA), violaxanthin, were also found with higherlevels of O₃ (80 nl l^-1). Levels of ABA itself were significantlyincreased by low level O₃ fumigation (30 nl l^-1) but significantlydecreased by exposure to 80 nl l^-1 O₃. This may indicate thathigher levels of atmospheric O₃ inhibit the final stages ofABA biosynthesis from violaxanthin.Copyright 1994, 1999 AcademicPress Abscisic acid, nitrogen assimilation, nitrogen fixation, ozone pollution, polyamines, violaxanthin     

Label-Free Enrichment of Adrenal Cortical Progenitor Cells Using Inertial Microfluidics

Passive and label-free isolation of viable target cells based on intrinsic biophysical cellular properties would allow for cost savings in applications where molecular biomarkers are known as well as potentially enable the separation of cells with little-to-no known molecular biomarkers. We have demonstrated the purification of adrenal cortical progenitor cells from digestions of murine adrenal glands utilizing hydrodynamic inertial lift forces that single cells and multicellular clusters differentially experience as they flow through a microchannel. Fluorescence staining, along with gene expression measurements, confirmed that populations of cells collected in different outlets were distinct from one another. Furthermore, primary murine cells processed through the device remained highly viable and could be cultured for 10 days in vitro. The proposed target cell isolation technique can provide a practical means to collect significant quantities of viable intact cells required to translate stem cell biology to regenerative medicine in a simple label-free manner.     

Isolation and characterization of an unprocessed extracellular myeloperoxidase in HL-60 cell cultures

Extracellular myeloperoxidase of human myeloid leukemia HL-60 cells was purified to homogeneity from its culture supernatant by ammonium sulfate fractionation, CM-Sepharose column chromatography, and monoclonal antibody-Sepharose affinity column chromatography. The yield of enzyme activity was 38% that of the ammonium sulfate fraction. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified preparation gave a single band of approximately 84 kDa. Analysis of protein blot with antibodies specific for the light and heavy chains of myeloperoxidase indicated that the enzyme contained a light and a heavy chain in a single polypeptide. The amino-terminal amino acid sequence of the enzyme began at amino acid residue 155 of the 745-amino acid sequence predicted from myeloperoxidase cDNA, indicating that the enzyme consisted of 591 amino acids. Sucrose density gradient centrifugation of the enzyme showed that the enzyme was a monomeric form. In pulse-chase experiments on HL-60 cells with [35S]methionine, pulse-labeled myeloperoxidase precursors were shown to be processed to a light chain and a heavy chain of cellular enzyme. During a 3-day chase period, newly formed processed monomeric enzyme was converted to a dimeric form.