Variation in storage alpha-glucans of the Porphyridiales (Rhodophyta)

Storage glucans were analyzed in the Porphyridiales which include the most primitive and phylogenetically diverged species in the Rhodophyta, to understand early evolution of the glucan structure in the Rhodophyta. The storage glucans of both Galdieria sulphuraria and Cyanidium caldarium consisted of glycogen, while those of Rhodosorus marinus, Porphyridium purpureum, P. sordidum and Rhodella violacea could be defined as semi-amylopectin. X-ray diffraction analysis of the glucans demonstrated variation in the crystalline structure: the patterns in P. purpureum and R. violacea were of A- and B-types, respectively, while alpha-glucans of R. marinus and P. sordidum displayed structures with lower crystallinity. Electron microscopic observations indicated that the alpha-glucans of P. sordidum consisted of two kinds of granules; a minor component of more dense granules with crystalline leaflets and a major component of softer ones without crystalline structure. Gel permeation chromatography showed that all the species containing the semi-amylopectin-type glucans also contained amylose, although the relative amounts of this fraction were different depending on the species. Our results are consistent with two distinct evolution scenarios defined either by the independent acquisition of semi-crystalline starch-like structures in the different plant lineages or more probably by the loss of starch and reversion to glycogen synthesis in cyanidian algae growing in hot and acid environments.     

Antagonistic jacalin-related lectins regulate the size of ER body-type beta-glucosidase complexes in Arabidopsis thaliana

PYK10/BGLU23 is a beta-glucosidase that is a major protein of ER bodies, which are endoplasmic reticulum (ER)-derived organelles that may be involved in defense systems. PYK10 has active and inactive forms. Active PYK10 molecules form large complexes with diameters ranging from 0.65 microm to > 70 microm. We identified three beta-glucosidases (PYK10, BGLU21 and BGLU22), five jacalin-related lectins (JALs) and a GDSL lipase-like protein (GLL) in the purified PYK10 complex. Expression levels of JALs and GLLs were lower in the nai1-1 mutant, which has no ER bodies, than in Col-0. The subcellular localization of PYK10 is predicted to be different from the localizations of JALs and GLLs. This suggests that PYK10 interacts with its partners (JALs and GLLs) when the subcellular structure is destroyed by pathogens. The PYK10 complex was found to be larger in the pbp1-1 and jal22-1 mutants than in Col-0, while it was smaller in the jal23-1, jal31-1 and jal31-2 mutants than in Col-0. These results show that two types of JALs having opposite roles regulate the size of the PYK10 complex antagonistically. We define the two types of lectins as a 'polymerizer-type lectin' and an 'inhibitor-type lectin'. Interestingly, the closest homologs of polymerizer-type lectins (JAL31 and JAL23) were inhibitor-type lectins (PBP1/JAL30 and JAL22). The pairs of polymerizer-type and inhibitor-type lectins reported here are good examples of genes that have evolved new functions after gene duplication (neofunctionalization).     

Removal of gelatin from live vaccines and DTaP—an ultimate solution for vaccine-related gelatin allergy

From the early 1990s infants started to receive acellular pertussis vaccine combined with diphtheria and tetanus toxoids (DTaP) before live vaccines such as measles, rubella, and mumps vaccines, which contained gelatin as a stabilizer. Then, an increasing number of cases of anaphylactic/allergic reactions to those live vaccines were reported. Almost all these cases had a previous history of receiving three or four doses of DTaP containing gelatin.Anaphylactic/allergic reactions to live measles vaccine were analyzed using information obtained from the Reporting System, a retrospective study, as well as from the Monitoring System, a prospective study. Dramatic decreases in anaphylactic/allergic reactions to live measles vaccines were observed immediately after each manufacturer marketed gelatin-free or gelatin (hypo-allergic)-containing live measles vaccine, and since the end of 1998 reports on anaphylactic/allergic reactions to live measles vaccine have almost ceased.     

Arabidopsis dynamin-related proteins DRP3A and DRP3B are functionally redundant in mitochondrial fission, but have distinct roles in peroxisomal fission

Two similar Arabidopsis dynamin-related proteins, DRP3A and DRP3B, are thought to be key factors in both mitochondrial and peroxisomal fission. However, the functional and genetic relationships between DRP3A and DRP3B have not been fully investigated. In a yeast two-hybrid assay, DRP3A and DRP3B interacted with themselves and with each other. DRP3A and DRP3B localized to mitochondria and peroxisomes, and co-localized with each other in leaf epidermal cells. In two T-DNA insertion mutants, drp3a and drp3b , the mitochondria are a little longer and fewer in number than those in the wild-type cells. In the double mutant, drp3a/drp3b , mitochondria are connected to each other, resulting in massive elongation. Overexpression of either DRP3A or DRP3B in drp3a/drp3b restored the particle shape of mitochondria, suggesting that DRP3A and DRP3B are functionally redundant in mitochondrial fission. In the case of peroxisomal fission, DRP3A and DRP3B appear to have different functions: peroxisomes in drp3a were larger and fewer in number than those in the wild type, whereas peroxisomes in drp3b were as large and as numerous as those in the wild type, and peroxisomes in drp3a/drp3b were as large and as numerous as those in drp3a . Although overexpression of DRP3A in drp3a/drp3b restored the shape and number of peroxisomes, overexpression of DRP3B did not restore the phenotypes, and often caused elongation instead. These results suggest that DRP3B and DRP3A have redundant molecular functions in mitochondrial fission, whereas DRP3B has a minor role in peroxisomal fission that is distinct from that of DRP3A.     

Peroxisomes Are Required for in Vivo Nitric Oxide Accumulation in the Cytosol following Salinity Stress of Arabidopsis Plants

Peroxisomes are unique organelles involved in multiple cellular metabolic pathways. Nitric oxide (NO) is a free radical active in many physiological functions under normal and stress conditions. Using Arabidopsis (Arabidopsis thaliana) wild type and mutants expressing green fluorescent protein through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo, this study analyzes the temporal and cell distribution of NO during the development of 3-, 5-, 8-, and 11-d-old Arabidopsis seedlings and shows that Arabidopsis peroxisomes accumulate NO in vivo. Pharmacological analyses using nitric oxide synthase (NOS) inhibitors detected the presence of putative calcium-dependent NOS activity. Furthermore, peroxins Pex12 and Pex13 appear to be involved in transporting the putative NOS protein to peroxisomes, since pex12 and pex13 mutants, which are defective in PTS1- and PTS2-dependent protein transport to peroxisomes, registered lower NO content. Additionally, we show that under salinity stress (100 mm NaCl), peroxisomes are required for NO accumulation in the cytosol, thereby participating in the generation of peroxynitrite (ONOO⁻) and in increasing protein tyrosine nitration, which is a marker of nitrosative stress.Peroxisomes are single membrane-bound organelles whose basic enzymatic constituents are catalase and H₂O₂-producing flavin oxidases as their basic enzymatic and are found in virtually all eukaryotic cell types (Corpas et al., 2001; Hayashi and Nishimura, 2006; Reumann et al., 2007; Pracharoenwattana and Smith, 2008; Palma et al., 2009). These oxidative organelles are characterized by metabolic plasticity, as their enzymatic content can vary according to the organism, cell/tissue type, and environmental conditions (Mullen et al., 2001; Hayashi and Nishimura, 2003; Corpas et al., 2009a). In higher plants, peroxisomes contain a complex battery of antioxidative enzymes, such as catalase, superoxide dismutase, the components of the ascorbate-glutathione cycle, and the NADP-dehydrogenases of the pentose-P pathway (Corpas et al., 2009a). The generation of superoxide radicals has also been reported in the matrices and membranes of peroxisomes (López-Huertas et al., 1999; del Río et al., 2006). All these findings point to the important role played by peroxisomes in the cellular metabolism of reactive oxygen species (Corpas et al., 2001, 2009a; del Río et al., 2006).Nitric oxide (NO) is a free radical involved in many physiological functions under normal and stress conditions in both animal and plant cells (Arasimowicz and Floryszak-Wieczorek, 2007; Corpas et al., 2007a, 2008; Neill et al., 2008). Unlike animal systems, knowledge of NO generation and subcellular location in plants remains largely elusive, and the data are sometimes contradictory and ambiguous (Zemojtel et al., 2006; Jasid et al., 2006; Gas et al., 2009). In previous studies, we detected l-Arg-dependent nitric oxide synthase (NOS) activity in isolated pea (Pisum sativum) leaf peroxisomes (Barroso et al., 1999). In a later study, using electron paramagnetic resonance techniques, we demonstrated the presence of NO in these types of peroxisomes (Corpas et al., 2004). However, several issues, such as whether NO is released into the cytosol and the physiological function of this free radical, remain unresolved.In this study, we provide an in vivo demonstration that Arabidopsis peroxisomes are essential for NO accumulation in the cytosol, thus participating in the generation of nitrosative stress under salinity conditions. In addition, using Arabidopsis mutants pex12 and pex13, we also suggest that these peroxins are involved in importing into peroxisomes the enzyme responsible for NO generation.     

Control of fibroblast growth factor (FGF) 7- and FGF1-induced mitogenesis and downstream signaling by distinct heparin octasaccharide motifs

Variation in length, disaccharide composition, and sulfation of heparan sulfate (HS) affects fibroblast growth factor (FGF) signaling. However, it is unclear whether the specific distribution of groups within oligosaccharides or random variations in charge density underlies the effects. Recently we showed that a mixture of undersulfated octasaccharides exhibiting 7 and 8 sulfates (7,8-S-OctaF7) generated from heparin had the highest affinity for FGF7 monitored by salt resistance (>0.60 M salt) of octasaccharide-FGF7 complexes. 7,8-S-OctaF7 also had the highest specific activity for formation of a complex with dimeric FGFR2IIIb competent to bind FGF7. Here we show that when endogenous HS was inhibited by chlorate treatment, 7,8-S-OctaF7 specifically supported FGF7-stimulated DNA synthesis and downstream signaling in FGFR2IIIb-expressing mouse keratinocytes. It failed to support FGF1 signaling in both HS-deficient mouse keratinocytes and 3T3 fibroblasts. In contrast, abundant, more highly sulfated and heterogenous mixtures of octasaccharides with lower affinity (0.30-0.60 M salt) for FGF7 supported FGF1-induced signaling in both cell types. In contrast to the two-component 7,8-S-OctaF7 mixture from FGF7, the high affinity octasaccharide fraction from FGF1 was a heterogeneous mixture with components ranging from 8 to 12 sulfates with 11-S-octasaccharides the most abundant. The high affinity fraction exhibited similar properties to the lower affinity fractions from both FGF1 and FGF7. Octasaccharide mixtures eluting from FGF1 between 0.30 and 0.60 M and above 0.60 M salt were nearly equal in support of FGF1 signaling in fibroblasts and keratinocytes. Both were deficient in support of FGF7-induced signaling in keratinocytes. The results show that both variations in overall charge density and specific distribution of charged groups within HS motifs exhibit FGF-specific control over formation of FGF-HS-FGFR complexes and downstream signaling.     

Manganese(II) complexes of an acyclic phenol-based dinucleating ligand with four methoxyethyl chelating arms: synthesis, structure, magnetism and electrochemistry

Dinuclear manganese(II) complexes [Mn₂(bomp)(PhCO₂)₂]BPh₄ (1), [Mn₂(bomp)(MeCO₂)₂]BPh₄ (2), and [Mn₂(bomp)(PhCO₂)₂]PF₆ (3) were synthesized with a dinucleating ligand 2,6-bis[bis(2-methoxyethyl)aminomethyl]-4-methylphenol [H(bomp)]. Dinuclear zinc complex [Zn₂(bomp)(PhCO₂)₂]PF₆ (4) was also synthesized for the purpose of comparison. X-ray analysis revealed that the complex 1·CHCl₃ contains two manganese ions bridged by the phenolic oxygen and two benzoate groups, forming a μ-phenoxo-bis(μ-benzoato)dimanganese(II) core. Magnetic susceptibility measurements of 1–3 over the temperature range 1.8–300 K indicated antiferromagnetic interaction (J=−4 to −6 cm⁻¹). Cyclic voltammograms of 3 showed a quasi-reversible oxidation process at +0.9 V versus a saturated sodium chloride calomel reference electrode, assigned to Mn^IIMn^II/Mn^IIMn^III.     

Allozyme diversity ofPittosporum (Pittosporaceae) on the Bonin (Ogasawara) Islands

The genusPittosporum includes about 160 species. Four species ofPittosporum occur in the Bonin Islands, and all of these are endemic to the islands. Electrophoretic studies of the four endemic species,P. tobira, from the Japanese mainland, andP. lutchuense var.denudatum from the Ryukyu Islands, were used to determine the origin and speciation pattern of the endemic species. 259 individuals were sampled from ten populations. Twenty loci in nine enzyme systems were resolved and used to calculate the gene frequencies for each population. A low genetic diversity was observed in three of the Bonin Island species, as is reported for other oceanic island plants. The exception,P. boninense, has the largest population size and widest distribution. A dendrogram generated by the UPGMA method shows two clusters. One consists of only the Bonin endemics, suggesting a monophyletic origin for these species.     

Roles for the two-hybrid system in exploration of the yeast protein interactome

Comprehensive analysis of protein-protein interactions is a challenging endeavor of functional proteomics and has been best explored in the budding yeast. The yeast protein interactome analysis was achieved first by using the yeast two-hybrid system in a proteome-wide scale and next by large-scale mass spectrometric analysis of affinity-purified protein complexes. While these interaction data have led to a number of novel findings and the emergence of a single huge network containing thousands of proteins, they suffer many false signals and fall short of grasping the entire interactome. Thus, continuous efforts are necessary in both bioinformatics and experimentation to fully exploit these data and to proceed another step forward to the goal. Computational tools to integrate existing biological knowledge buried in literature and various functional genomic data with the interactome data are required for biological interpretation of the huge protein interaction network. Novel experimental methods have to be developed to detect weak, transient interactions involving low abundance proteins as well as to obtain clues to the biological role for each interaction. Since the yeast two-hybrid system can be used for the mapping of the interaction domains and the isolation of interaction-defective mutants, it would serve as a technical basis for the latter purpose, thereby playing another important role in the next phase of protein interactome research.