Biosynthesis of Leaf Alcohol: Kinetic Study of Lipoxygenase Activation Process Caused by Hydroperoxylinoleic Acid

Quaternary structure of ribulose-1, 5-bisphosphate (RuP₂) carboxylase from the autotrophically grown cells of blue-green alga, Anabaena cylindrica, was studied. Sedimentation coefficient (s_{20, w}) of the enzyme was determined to be 18.3 S by the sucrose density gradient centrifugation. The molecular weight was estimated to be 5.0 × 10⁵ by the Sepharose 4B gel filtration technique. The purification of the enzyme from the algal cells was undertaken by means of sucrose density gradient centrifugation and DEAE-Sephadex A–50 ion-exchange column chromatography, and the structural make-up of the enzyme containing two subunits, A (M. W., 5.2 × 10⁴) and B (M. W., 1.2 × 10⁴) was established by the Na-dodecylsulfate polyacrylamide gel electrophoresis experiment. Structural similarity of the algal RuP₂carboxylase with the spinach enzyme was further demonstrated by the Ouchterlony double immunodiffusion experiment.     

Identification of the minimum region of Bordetella pertussis Vag8 required for interaction with C1 inhibitor

An autotransporter of Bordetella pertussis, virulence-associated gene 8 (Vag8), binds and inactivates the complement regulator, C1 inhibitor (C1-Inh), and plays a role in evasion of the complement system. However, the molecular interaction between Vag8 and C1-Inh remains unclear. Here, we localized the minimum region of Vag8 required for interaction with C1-Inh by examining the differently truncated Vag8 derivatives for the ability to bind and inactivate C1-Inh. The truncated Vag8 containing amino-acid residues 102–548, but not 102–479 and 202–648, showed the full activity of intact Vag8, suggesting that the separate 102–202 and 548–648 amino-acid regions of Vag8 mediate the interaction with C1-Inh.     

Cellular localization of mitotic RAD21 with repetitive amino acid motifs in Allium cepa

Onion can be used in experimental observation of mitotic cell division in plant science because its chromosome is large and easy to observe. However, molecular genetic studies are difficult in onion because of its large genome size, and only limited information of onion genes has been available to date. Here we cloned and characterized an onion homologue of mitotic RAD21 gene, AcRAD21-1, to develop a molecular marker of mitosis. The N-terminal, middle, and C-terminal regions of deduced AcRAD21-1 protein sequence were conserved with Arabidopsis SYN4/AtRAD21.3 and rice OsRAD21-1, whereas three characteristic types of repetitive motifs (Repeat-1, Repeat-2/2′, and Repeat-3) were observed between the conserved regions. Such inserted repetitive amino acid sequences enlarge the AcRAD21-1 protein into almost 200 kDa, which belongs to the largest class of plant proteins. Genomic organization of the AcRAD21-1 locus was also determined, and the possibility of tandem exon duplication in Repeat-2 was revealed. Subsequently, the polyclonal antiserum was raised against the N-terminal region of AcRAD21-1, and purified by affinity chromatography. Immunohistochemical analysis with the purified antibody successfully showed localization of AcRAD21-1 in onion mitosis, suggesting that it can be used as a molecular marker visualizing dynamic movement of cohesin.     

Modulation of Hepatocarcinoma Cell Morphology and Activity by Parylene-C Coating on PDMS

Background

The ability to understand and locally control the morphogenesis of mammalian cells is a fundamental objective of cell and developmental biology as well as tissue engineering research. We present parylene-C (ParC) deposited on polydimethylsiloxane (PDMS) as a new substratum for in vitro advanced cell culture in the case of Human Hepatocarcinoma (HepG2) cells.

Principal Findings

Our findings establish that the intrinsic properties of ParC-coated PDMS (ParC/PDMS) influence and modulate initial extracellular matrix (ECM; here, type-I collagen) surface architecture, as compared to non-coated PDMS substratum. Morphological changes induced by the presence of ParC on PDMS were shown to directly affect liver cell metabolic activity and the expression of transmembrane receptors implicated in cell adhesion and cell-cell interaction. These changes were characterized by atomic force microscopy (AFM), which elucidated differences in HepG2 cell adhesion, spreading, and reorganization into two- or three-dimensional structures by neosynthesis of ECM components. Local modulation of cell aggregation was successfully performed using ParC/PDMS micropatterns constructed by simple microfabrication.

Conclusion/Significance

We demonstrated for the first time the modulation of HepG2 cells'' behavior in relation to the intrinsic physical properties of PDMS and ParC, enabling the local modulation of cell spreading in a 2D or 3D manner by simple microfabrication techniques. This work will provide promising insights into the development of cell-based platforms that have many applications in the field of in vitro liver tissue engineering, pharmacology and therapeutics.     

Crystal structure of oxidized cytochrome c(6A) from Arabidopsis thaliana

Compared with algal and cyanobacterial cytochrome c(6), cytochrome c(6A) from higher plants contains an additional loop of 12 amino acid residues. We have determined the first crystal structure of cytochrome c(6A) from Arabidopsis thaliana at 1.5 Angstrom resolution in order to help elucidate its function. The overall structure of cytochrome c(6A) follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys16 and Cys19, and the iron has octahedral coordination with His20 and Met60 as the axial ligands. Two cysteine residues (Cys67 and Cys73) within the characteristic 12 amino acids loop form a disulfide bond, contributing to the structural stability of cytochrome c(6A). Our model provides a chemical basis for the known low redox potential of cytochrome c(6A) which makes it an unsuitable electron carrier between cytochrome b(6)f and PSI.     

Thermonasty of young main stems of Phryma leptostachya (Phrymaceae)

Thermonasty-like responses were observed on some main stems of Phryma leptostachya (Phrymaceae) plants cultivated in the open. To confirm the thermonastic nature of these responses, the plants were moved into an artificially illuminated chamber and observed under controlled conditions. At low temperatures (about 12°C) and in the dark, slanted terrestrial young main stems of P. leptostachya became prostrate. At higher temperatures (about 25°C) and in the dark, the prostrate stems became slanted. Their movement was confirmed under controlled conditions and is therefore considered to be thermonastic in nature.     

Phylogenetic and morphological diversity of Bacteroidales members associated with the gut wall of termites

The microbial community adherent directly or indirectly to the gut wall of termites is distinct from that of the other habitats in the gut. The bacterial 16S rRNA genes were identified from the fractionated gut walls of two termite species, Hodotermopsis sjoestedti and Neotermes koshunensis, and compared with those previously identified from Reticulitermes speratus. Surprisingly, the bacterial constituents were almost entirely different among the termites at the phylotype level (the criterion of the phylotype was >97% nucleotide identity). Bacteria in the order Bacteroidales, which were commonly abundant symbionts on gut walls, were phylogenetically analyzed. They were dispersed in a number of clusters formed by phylotypes from the guts of various termites. In situ hybridization with probes specific for some phylotypes and a phylogenetic cluster detected the cells of several Bacteroidales members with a significant variety of cell morphology in the gut wall fractions, which reflects the phylogenetic diversity of this order.