Relationships Among Stop Codon Usage Bias, Its Context, Isochores, and Gene Expression Level in Various Eukaryotes

It is well known that stop codons play a critical role in the process of protein synthesis. However, little effort has been made to investigate whether stop codon usage exhibits biases, such as widely seen for synonymous codon usage. Here we systematically investigate stop codon usage bias in various eukaryotes as well as its relationships with its context, GC3 content, gene expression level, and secondary structure. The results show that there is a strong bias for stop codon usage in different eukaryotes, i.e., UAA is overrepresented in the lower eukaryotes, UGA is overrepresented in the higher eukaryotes, and UAG is least used in all eukaryotes. Different conserved patterns for each stop codon in different eukaryotic classes are found based on information content and logo analysis. GC3 contents increase with increasing complexity of organisms. Secondary structure prediction revealed that UAA is generally associated with loop structures, whereas UGA is more uniformly present in loop and stem structures, i.e., UGA is less biased toward having a particular structure. The stop codon usage bias, however, shows no significant relationship with GC3 content and gene expression level in individual eukaryotes. The results indicate that genomic complexity and GC3 content might contribute to stop codon usage bias in different eukaryotes. Our results indicate that stop codons, like synonymous codons, exhibit biases in usage. Additional work will be needed to understand the causes of these biases and their relationship to the mechanism of protein termination. [Reviewing Editor: Dr. Manyuan Long]     

Enhanced anti-angiogenic effect of a deletion mutant of plasminogen kringle 5 on neovascularization

Kringle 5 (K5), a proteolytic fragment of plasminogen, has been proved to be an angiogenic inhibitor. Previously, we have evaluated the effect of K5 on the vascular leakage and neovascularization in a rat model of oxygen-induced retinopathy. In this study, we expressed K5 and a deletion mutant of K5 (K5 mutant) in a prokaryocyte expression system and purified them by affinity chromatography. K5 mutant was generated by deleting 11 amino acids from K5 while retaining the three disulfide bonds. The anti-angiogenic activity of intact K5 and K5 mutant were compared in endothelial cells and retinal neovascularization rat model. K5 mutant inhibited the proliferation of primary human retinal capillary endothelial cells (HRCEC) in a concentration-dependent manner, with an apparent EC50 of approximate 35 nmol/L, which is twofold more potent than intact K5. In the even higher concentration range, K5 mutant did not inhibit pericytes from the same origin of HRCEC, which suggested an endothelial cell-specific inhibition. K5 mutant had no effect on normal liver cells and Bel7402 hepatoma cells even at high concentration range either. Intravitreal injection of the K5 and mutant in the oxygen-induced retinopathy rat model both resulted in significantly fewer neovascular tufts and nonperfusion area than controls with PBS injection, as shown by fluorescein angiography. Furthermore, K5 mutant exhibited more strong inhibition effect on neovascularization than intact K5 by quantification of vascular cells. These results suggest that this K5 deletion mutant is a more potent angiogenic inhibitor than intact K5 and may have therapeutic potential in the treatment of those disorders with neovascularization, such as solid tumor, diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, and hyperplasia of prostate.     

A trifluoromethyl substituted organoimido derivative of the hexametalate cluster: synthesis, crystal structure and bioactivity of [Mo6O17(NAr)2]2- (Ar=o-CF3C6H4)

The hexamolybdate cluster has been functionalized with fluorine-containing aromatic amine by the DCC (N,N'-dicyclohexylcarbodiimide) dehydrating protocol and a novel bifunctionalized arylimido derivative of hexamolybdate bearing the electron-withdrawing trifluoromethyl group, (Bu(4)N)(2)[Mo(6)O(17)(NAr)(2)] (Ar=o-CF(3)C(6)H(4)), has been prepared. Complete assignments were achieved for the title compound by elemental analysis, IR, (1)H NMR, UV/visible and single-crystal X-ray diffraction analyses. The preliminary agricultural biological activity test indicated that it has some herbicidal and insecticidal activities.     

Phylogenetics and evolution of nematode-trapping fungi (Orbiliales) estimated from nuclear and protein coding genes

The systematic classification of nematode-trapping fungi is redefined based on phylogenies inferred from sequence analyses of 28S rDNA, 5.8S rDNA and beta-tubulin genes. Molecular data were analyzed with maximum parsimony, maximum likelihood and Bayesian analysis. An emended generic concept of nematode-trapping fungi is provided. Arthrobotrys is characterized by adhesive networks, Dactylellina by adhesive knobs, and Drechslerella by constricting-rings. Phylogenetic placement of taxa characterized by stalked adhesive knobs and non-constricting rings also is confirmed in Dactylellina. Species that produce unstalked adhesive knobs that grow out to form loops are transferred from Gamsylella to Dactylellina, and those that produce unstalked adhesive knobs that grow out to form networks are transferred from Gamsylella to Arthrobotrys. Gamsylella as currently circumscribed cannot be treated as a valid genus. A hypothesis for the evolution of trapping-devices is presented based on multiple gene data and morphological studies. Predatory and nonpredatory fungi appear to have been derived from nonpredatory members of Orbilia. The adhesive knob is considered to be the ancestral type of trapping device from which constricting rings and networks were derived via two pathways. In the first pathway adhesive knobs retained their adhesive material forming simple two-dimension networks, eventually forming complex three-dimension networks. In the second pathway adhesive knobs lost their adhesive materials, with their ends meeting to form nonconstricting rings and they in turn formed constricting rings with three inflated-cells.     

Exogenous ethylene influences flower opening of cut roses (<Emphasis Type="Italic">Rosa hybrida</Emphasis>) by regulating the genes encoding ethylene biosynthesis enzymes

The purpose of this paper is to investigate the differential responses of flower opening to ethylene in two cut rose cultivars, ‘Samantha’, whose opening process is promoted, and ‘Kardinal’, whose opening process is inhibited by ethylene. Ethylene production and 1-aminocyclopropane-1-carboxylate (ACC) synthase and oxidase activities were determined first. After ethylene treatment, ethylene production, ACC synthase (ACS) and ACC oxidase (ACO) activities in petals increased and peaked at the earlier stage (stage 3) in ‘Samantha’, and they were much more dramatically enhanced and peaked at the later stage (stage 4) in ‘Kardinal’ than control during vasing. cDNA fragments of three Rh-ACSs and one Rh-ACO genes were cloned and designated as Rh-ACS1, Rh-ACS2, Rh-ACS3 and Rh-ACO1 respectively. Northern blotting analysis revealed that, among three genes of ACS, ethylene-induced expression patterns of Rh-ACS3 gene corresponded to ACS activity and ethylene production in both cultivars. A more dramatic accumulation of Rh-ACS3 mRNA was induced by ethylene in ‘Kardinal’ than that of ‘Samantha’. As an ethylene action inhibitor, STS at concentration of 0.2 mmol/L generally inhibited the expression of Rh-ACSs and Rh-ACO in both cultivars, although it induced the expression of Rh-ACS3 transiently in ‘Kardinal’. Our results suggests that ‘Kardinal’ is more sensitive to ethylene than ‘Samantha’; and the changes of Rh-ACS3 expression caused by ethylene might be related to the acceleration of flower opening in ‘Samantha’ and the inhibition in ‘Kardinal’. Additional results indicated that three Rh-ACSs genes were differentially associated with flower opening and senescence as well as wounding.     

Coadministration of interleukin 2 plasmid DNA with combined DNA vaccines significantly enhances the protective efficacy against Mycobacterium tuberculosis

Coadministration of interleukin 2(IL-2) plasmid DNA with combined DNA vaccines enhanced Th1-type cellular responses by producing higher amounts of IFN-gamma with a higher ratio of antigen-specific IgG2a/IgG1. The IFN-gamma specific for Ag85B, MPT64, and MPT83 in this group was 415, 267, and 255 U/ml, respectively, and was 1.6-, 1.8-, and 2.5-fold higher than that of the same vaccine without adding IL-2. The IgG2a/IgG1 ratio for Ag85B, MPT64, and MPT83 was 4, 8, and 4, respectively, upon addition of the genetic adjuvant in the DNA vaccine, which was four times higher for every antigen when IL-2 was not included. Fluorescence activated cell sorter (FACS) analysis showed that, in the presence of IL-2, CD8+ and CD4+ T cells increased significantly, whereas in the absence of the genetic adjuvant, only a mild increase was observed for CD8+ T cells compared to the vector DNA-treated group. Bacterial CFU was reduced to less than 1/100 in the lung and to about 1/10 in the spleen relative to the same combined DNA vaccine without IL-2. The lungs of this group of mice showed much less damage due to an influx of epithelioid macrophages and less lymphocytes. RT-PCR showed that antigen genes could be detected in more organs and for a longer period of time when treated with combined DNA vaccine formulated in IL-2. We suggest that IL-2 enhanced the immunigencity and protective efficacy in immunized mice by improving the Th1-type response and also by prolonging the antigen gene expression in different organs.     

Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex

We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-gamma1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase alpha1 subunit interacts with PLC-gamma1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma1 and IP3 receptors together to form a Ca(2+)-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma1 and IP3R2 coimmunoprecipitated with the Na/K-ATPase and ouabain increased this interaction in a dose- and time-dependent manner in LLC-PK1 cells. Depletion of cholesterol abolished the effects of ouabain on this interaction. Second, ouabain induced phosphorylation of PLC-gamma1 at Tyr(783) and activated PLC-gamma1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca(2+) release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-gamma1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca(2+).     

ERK phosphorylates p66shcA on Ser36 and subsequently regulates p27kip1 expression via the Akt-FOXO3a pathway: implication of p27kip1 in cell response to oxidative stress

Mice deficient for p66shcA represent an animal model to link oxidative stress and aging. p66shcA is implicated in oxidative stress response and mitogenic signaling. Phosphorylation of p66shcA on Ser36 is critical for its function in oxidative stress response. Here we report the identification of ERK as the kinase phosphorylating p66shcA on Ser36. Activation of ERKs was necessary and sufficient for Ser36 phosphorylation. p66shcA interacted with ERK and was demonstrated to be a substrate for ERK, with Ser36 being the major phosphorylation site. Furthermore, in response to H2O2, inhibition of ERK activation repressed p66shcA-dependent phosphorylation of FOXO3a and the down-regulation of its target gene p27kip1. Down-regulation of p27 might promote cell survival, as p27 played a proapoptotic role in oxidative stress response. As a feedback regulation, Ser36 phosphorylated p66shcA attenuated H2O2-induced ERK activation, whereas p52/46shcA facilitated ERK activation, which required tyrosine phosphorylation of CH1 domain. p66shcA formed a complex with p52/46ShcA, which may provide a platform for efficient signal propagation. Taken together, the data suggest there exists an interplay between ERK and ShcA proteins, which modulates the expression of p27 and cell response to oxidative stress.     

Prk1p

The protein kinase Prk1p (standing for p53 regulating kinase 1) of the yeast Saccharomyces cerevisiae is the prototype of a kinase family identified recently as important regulators of the actin cytoskeleton and endocytosis. These kinases all have a highly homologous serine/threonine kinase domain in their N-terminal region but share no significant homology in other regions. Prk1p also contains a proline-rich motif near its C-terminus that is required for the proper subcellular localization of the protein. The kinase activity of Prk1p has been confirmed by both in vitro and in vivo studies and shown to be essential for the protein's function. To date, several proteins that play essential roles in actin cytoskeleton organization and endocytosis have been identified as the regulatory targets of Prk1p. Phosphorylation on the [L/I/V/N]xx[Q/N/T/S]xTG motifs by Prk1p results in a down-regulation of the functions of these target proteins. The observation that many yeast proteins involved in the actin cytoskeleton organization and endocytosis contain the Prk1p phosphorylation motifs has led to the hypothesis that the Prk1p family of kinases are possibly the general regulators of the actin cytoskeleton and endocytosis in yeast.