Cloning and Sequence Analysis of Genes for Cyclophilin from Arabidopsis thaliana

Two genomic DNA clones encoding cyclophilin (CyP) from Arabidopsisthaliana were isolated. The deduced protein products of thesegenes appeared to be cytosol-localized isoforms given the absenceof a specific presequence for targeting to cellular compartments.Thus, multiple CyPs may exist and function in the cytosol ofArabidopsis thaliana. ⁴Present address: Division 1 of Gene Expression and Regulation,National Institute for Basic Biology, Okazaki, 444 Japan     

Sensitive ultraviolet monitoring of aldoses in automated borate complex anion-exchange chromatography with 2-cyanoacetamide

A convenient method for postcolumn carbohydrate labeling has been developed. Eluates of borate complex anion-exchange columns are mixed with a reagent solution prepared from an aqueous solution of 2-cyanoacetamide and a borate buffer (pH 10.5), and the mixture is heated in a 10-m reaction coil at 100°C. Measurement of the absorbance of the product at 276 nm permits high reproducibility determination of 5 to 500 nmol of aldoses. Some carbonyl compounds are positive to this reaction, but most do not interfere with the analysis because their peaks do not appear in the aldose region. Ascorbate gives a small peak between those of mannose and fucose, but interference is negligible for equimolar amounts of ascorbate and these aldoses. This method is applied to and gives satisfactory results in the analysis of monosaccharides from various types of glycoconjugates.     

Precolumn labeling of reducing carbohydrates with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone: analysis of neutral and sialic acid-containing oligosaccharides found in glycoproteins.

A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.     

Expression and possible role of PVR/CD155/Necl-5 in osteoclastogenesis

Osteoclasts, the bone-resorbing cells, are differentiated from hematopoietic precursors via two-step cell–cell interactions. One is the interaction between the osteoclast precursor and the stromal cell to initiate differentiation. The other is the interaction among osteoclast precursors to form multinucleated osteoclasts. Recently, the poliovirus receptor (PVR, CD155, Necl-5) was reported to play important roles in cell adhesion and migration. However, there are no reports of PVR in osteoclastogenesis. In this paper, we examined the expression of PVR and its ligand, DNAX accessory molecule-1 (DNAM-1, CD226), in osteoclast precursors, mature osteoclasts, and stromal cells. We found that the PVR was constitutively expressed in both osteoclast cells and stromal cells. The expression of PVR was not changed at various stages of osteoclast formation. In contrast, the expression of DNAM-1 was observed in mononuclear cells and was down-regulated during osteoclastogenesis. Moreover, multinucleated osteoclast formation was inhibited by treatment with the extracellular domain of DNAM-1 (ED-DNAM-1) as a soluble ligand for PVR, but mononuclear preosteoclast formation was not affected. Especially, during the 7-day cultivation, osteoclast formation was suppressed by the treatment with ED-DNAM-1 on days 6 and 7, when the mononuclear preosteoclasts fused into multinucleated osteoclasts. This suppression was abrogated partially by a small interfering RNA specific for PVR. These results suggest that, at least in part, the binding of PVR with DNAM-1 negatively regulates osteoclast formation. Furthermore, our results indicate that the cellular fusion process may be inhibited by the PVR-mediated signaling.     

Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants

We made a series of improved Gateway binary vectors (pGWBs) for plant transformation. Fifteen different reporters and tags, sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7, and TAP, were employed. Some vectors carry the 2x35S-Omega promoter for higher-level expression. The kanamycin- and hygromycin-resistant markers are independently available for each of the 43 types of vectors, thus an additional transformation of once-transformed plants can be carried out easily. Their small size and high-copy number in Escherichia coli make possible easier handling at plasmid preparation and sequencing. Improved pGWBs should be a powerful tool for transgenic research in plants.     

A comparison of epiphytic diatom communities on <Emphasis Type="Italic">Plocamium cartilagineum</Emphasis> (Plocamiales,Florideophyceae) from two Antarctic areas

Our understanding of diatoms, one of the most important Antarctic primary producers, is based mostly on investigations of plankton, sea-ice, and sediment samples. Herein, we contribute to the limited research devoted to benthic Antarctic diatoms by presenting a study on epiphytic diatom communities sampled in two remote Antarctic regions: Admiralty Bay (maritime Antarctica, Antarctic Peninsula) and Terra Nova Bay (Ross Sea). Recent studies have demonstrated that the most critical factor for the local epiphytic diatom communities was the nature of the substrate. In order to eliminate this factor so we could evaluate other potential controls, we sampled epiphytic diatoms from only one substrate that is common to both regions: the macroalgae Plocamium cartilagineum (L.) Dixon. Thalli of P. cartilagineum and their associated microalgal community was collected in January 2011 (Admiralty Bay) and 2012 (Terra Nova Bay) from a water depth of 5–25 m. Dehydrated macroalgal pieces were placed on stubs and sputter-coated, which allowed observation of diatoms attached to the substrate in their original position using scanning electron microscopy. A total of 72 taxa were observed, of which 31 taxa were common to both regions. Cell abundance and diatom growth form dominance were significantly different in Admiralty Bay and Terra Nova Bay samples. Total diatom abundance was higher in Admiralty Bay samples, dominated by adnate diatoms (Cocconeis spp.), but the number of taxa found as well as the values of ecological indices were higher for samples from the Ross Sea, where motile forms were dominant (Navicula spp.). Our results suggest that Antarctic shallow-water benthic habitats may present a high degree of microniche heterogeneity and highlight the need of fine-scale analyses in microbial studies. We also suggest grazers as a factor that contributes greatly to the observed differences.