Destination-selective long-distance movement of phloem proteins

The phloem macromolecular transport system plays a pivotal role in plant growth and development. However, little information is available regarding whether the long-distance trafficking of macromolecules is a controlled process or passive movement. Here, we demonstrate the destination-selective long-distance trafficking of phloem proteins. Direct introduction, into rice (Oryza sativa), of phloem proteins from pumpkin (Cucurbita maxima) was used to screen for the capacity of specific proteins to move long distance in rice sieve tubes. In our system, shoot-ward translocation appeared to be passively carried by bulk flow. By contrast, root-ward movement of the phloem RNA binding proteins 16-kD C. maxima phloem protein 1 (CmPP16-1) and CmPP16-2 was selectively controlled. When CmPP16 proteins were purified, the root-ward movement of CmPP16-1 became inefficient, suggesting the presence of pumpkin phloem factors that are responsible for determining protein destination. Gel-filtration chromatography and immunoprecipitation showed that CmPP16-1 formed a complex with other phloem sap proteins. These interacting proteins positively regulated the root-ward movement of CmPP16-1. The same proteins interacted with CmPP16-2 as well and did not positively regulate its root-ward movement. Our data demonstrate that, in addition to passive bulk flow transport, a destination-selective process is involved in long-distance movement control, and the selective movement is regulated by protein-protein interaction in the phloem sap.     

Molecular characterization of woodchuck interleukin 15 (wIL-15) and detection of its expression in liver samples of woodchucks infected with woodchuck hepatitis virus (WHV)

Interleukin 15 (IL-15) is a member of the four-helix bundle cytokine family and has T cell growth factor activity. IL-15 plays a unique role in both innate and adaptive immune cell homeostasis, particularly for the development of NK cells and CD8+memory cells. It may be useful for stimulation of specific immune responses in chronic viral infection such as hepatitis B virus infection. The woodchuck model is an informative animal model for studies on hepadnavirus infection and therapeutic interventions. Here, the complete coding sequence of woodchuck IL-15 (wIL-15) was cloned and sequenced. wIL-15 shows a high homology (>70%) to its counterparts of other mammalian species. His-tagged recombinant wIL-15 protein was expressed and purified and showed the ability to promote the proliferation of activated mouse splenocytes and woodchuck peripheral blood lymphocytes. Further, examination of mRNA amounts in liver samples of woodchucks by semi-quantitative RT-PCR showed a slightly increased expression of wIL-15 in woodchuck livers during chronic woodchuck hepatitis virus infection. This available information will provide a basis for further studies on the function of IL-15 in the context of acute and chronic hepadnavirus infection and its potential therapeutic use for chronic hepatitis B virus infection in the woodchuck model.     

Peptidoglycan inducible expression of a serine proteinase homologue from kuruma shrimp (Marsupenaeus japonicus)

A cDNA encoding a serine proteinase homologue of kuruma shrimp (Marsupenaeus japonicus) was cloned. The 1257 bp cDNA encodes a 339 amino acid putative peptide, with a signal sequence of 16 amino acid residues. The deduced amino acid sequence is 42-67% similar to the immune-related serine proteinases and serine proteinase homologues of arthropods. It contains catalytic triad residues in the putative catalytic domain except for one substitution of Ser by a Gly residue. The six cysteine residues that form three disulphide bridges in most serine proteinases were conserved. The M. japonicus serine proteinase homologue was mainly expressed in haemocytes, in which expression dramatically increased after 3 days feeding with peptidoglycan at 0.2 mg kg(-1) shrimp body weight per day.     

Regression of abdominal aortic aneurysm by inhibition of c-Jun N-terminal kinase

Abdominal aortic aneurysm (AAA) is a common disease among elderly people that, when surgical treatment is inapplicable, results in progressive expansion and rupture of the aorta with high mortality. Although nonsurgical treatment for AAA is much awaited, few options are available because its molecular pathogenesis remains elusive. Here, we identify JNK as a proximal signaling molecule in the pathogenesis of AAA. Human AAA tissue showed a high level of phosphorylated JNK. We show that JNK programs a gene expression pattern in different cell types that cooperatively enhances the degradation of the extracellular matrix while suppressing biosynthetic enzymes of the extracellular matrix. Selective inhibition of JNK in vivo not only prevented the development of AAA but also caused regression of established AAA in two mouse models. Thus, JNK promotes abnormal extracellular matrix metabolism in the tissue of AAA and may represent a therapeutic target.     

Solution structure of the PWWP domain of the hepatoma-derived growth factor family

Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded beta-barrel with a PWWP-specific long loop connecting beta2 and beta3 (PR-loop), followed by a helical region including two alpha-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the beta1/beta2 loop (beta-beta arch) and the beta3/beta4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.     

Methodological considerations and approach to cross-technique comparisons using in vivo coronary plaque characterization based on intravascular ultrasound radiofrequency data analysis: insights from the Integrated Biomarker and Imaging Study (IBIS)

Grey scale intravascular ultrasound (IVUS) is a valuable clinical tool to assess the extent and severity of coronary atheroma. However, it cannot reliably identify plaques with a high-risk of future clinical events. Serial IVUS studies to assess the progression and/or regression of atherosclerotic plaques demonstrated only modest effects, of pharmacological intervention on plaque burden, even when clinical efficacy is documented. Spectral analysis of radiofrequency ultrasound data (IVUS-virtual histology (IVUS-VH), Volcano Therapeutics, Rancho Cordova, CA) has the potential to characterize accurately plaque composition. The Integrated Biomarker and Imaging Study (IBIS) evaluated both invasive and non-invasive imaging techniques along with the assessment of novel biomarkers to characterize sub-clinical atherosclerosis. IVUS-VH was not included at the start of the IBIS protocol. The purpose of this paper is to describe the methodology we used to obtain and analyse IVUS-VH images and the approach to cross-correlations with the other techniques.     

Synergistic effects of Glu130Asp substitution in the type II polyhydroxyalkanoate (PHA) synthase: enhancement of PHA production and alteration of polymer molecular weight

In vitro evolution of the polyhydroxyalkanoate (PHA) synthase gene from Pseudomonas sp. 61-3 (phaC1(Ps)) has been performed to generate highly active enzymes. In this study, a positive mutant of PHA synthase, Glu130Asp (E130D), was characterized in detail in vivo and in vitro. Recombinant Escherichia coli strain JM109 harboring the E130D mutant gene accumulated 10-fold higher (1.0 wt %) poly(3-hydroxybutyrate) [P(3HB)] from glucose, compared to recombinant E. coli harboring the wild-type PHA synthase gene (0.1 wt %). Recombinant E. coli strain LS5218 harboring the E130D PHA synthase gene grown on dodecanoate produced more poly(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] (20 wt %) copolymer than an LS5218 strain harboring the wild-type PHA synthase gene (13 wt %). The E130D mutation also resulted in the production of copolymer with a slight increase in 3HB composition, compared to copolymer produced by the wild-type PHA synthase. In vitro enzyme activities of the E130D PHA synthase toward various 3-hydroxyacyl-CoAs (4-10 carbons in length) were all higher than those of the wild-type enzyme. The combination of the E130D mutation with other beneficial mutations, such as Ser325Thr and Gln481Lys, exhibited a synergistic effect on in vivo PHA production and in vitro enzyme activity. Interestingly, gel-permeation chromatography analysis revealed that the E130D mutation also had a synergistic effect on the molecular weight of polymers produced in vivo.     

Promotion of evolution by intracellular coexistence of mutator and normal DNA polymerases

The efficient evolution of a population requires both genetic diversity and stable reproduction of advantageous genotypes. The accuracy of DNA replication guarantees the stable reproduction, while errors during DNA replication produce the genetic diversity. Thus, one key to the promotion of evolution is inherent in DNA replication. In bacteria, replication forks progress bidirectionally from the single origin of replication on a genome. One replication fork contains two DNA polymerase molecules so that four DNA polymerases simultaneously carry out the replication of a genome. It is generally believed that the fidelity of the intracellular DNA polymerases is identical (parity strategy). To test this, we examined the effects of the intracellular coexistence of a mutator polymerase with low fidelity and a normal polymerase with high fidelity on adaptive evolution (disparity strategy). From the analysis using genetic algorithms based on the bacterial replication, it was found that the population using the disparity strategy could further expand its genetic diversity and preserve the advantageous genotypes more profoundly than the parity population. This strongly suggests that bacteria replicating with a disparity strategy may undergo rapid evolution, particularly during severe environmental changes. The implications of the conspicuous adaptability of Escherichia coli mutator strains are discussed in this context.