Design and functional analysis of actomyosin motor domain chimera proteins

To gain more structural and functional information on the actomyosin complexes, we have engineered chimera proteins carrying the entire Dictyostelium actin in the loop 2 sequence of the motor domain of Dictyostelium myosin II. Although the chimera proteins were unable to polymerize by themselves, addition of skeletal actin promoted polymerization. Electron microscopic observation demonstrated that the chimera proteins were incorporated into actin filaments, when copolymerized with skeletal actin. Copolymerization with skeletal actin greatly enhanced the MgATPase, while the chimera proteins without added skeletal actin hydrolyzed ATP at a very low rate. These results indicate that the actin part and the motor domain part of the chimera proteins are correctly folded, but the chimera proteins are structurally stressed so that efficient polymerization is inhibited.     

Presence of free D-amino acids in microalgae

Several species of microalgae (phytoplankton), 4 species of freshwater algae and 4 species of marine diatoms, were cultured germ-free in the laboratory. The presence of free D-amino acids was verified in these species by a reversed-phase HPLC analysis. D-Aspartate was detected in all the microalgae examined, but D-alanine was only present in the marine diatoms. The D-amino acid content in Asterionella sp. of the marine diatoms increased from the exponential phase to the stationary phase and then decreased to the phase of decline.     

Detection of 1270 nm emission from singlet oxygen and photocytotoxic property of sugar-pendant 60 fullerenes

Sugar-pendant [60] fullerene derivatives have been prepared from carbohydrate-linked azides 1a-e. Both monosugar (4a-e) and bissugar derivatives (5a-e) produce singlet oxygen ((1)O(2)) under laser irradiation (355 nm) proved by the direct observation of (1)O(2) emission at 1270 nm. Monosugar derivatives exhibit photocytotoxicity varying by the attached sugar molecule.     

Evolutionary Studies on Uricases of Fungal Endosymbionts of Aphids and Planthoppers

Aphids belonging to the three genera Tuberaphis, Glyphinaphis, and Cerataphis contain extracellular fungal symbionts that resemble endocellular yeast-like symbionts of planthoppers. Whereas the symbiont of planthoppers has a uricase (urate oxidase; EC 1.7.3.3) and recycles uric acid that the host stores, no uric acid was found in Tuberaphis styraci, and its fungal symbiont did not exhibit the uricase activity. However, the fungal symbionts of these aphids, including that of T. styraci, were shown to have putative uricase genes, or pseudogenes, for the uricase. Sequence analysis of these genes revealed that deleterious mutations occurred independently on each lineage of Glyphinaphis and Tuberaphis, while no such mutation was found in the lineage of Cerataphis. These genes were almost identical to those cloned from the symbionts of planthoppers, though the host aphids and planthoppers are phylogenetically distant. To estimate the phylogenetic relationship in detail between the fungal symbionts of aphids and those of planthoppers, a gene tree was constructed based on the sequences of the uricase genes including their flanking regions. As a result, the symbionts of planthoppers and Tuberaphis aphids formed a sister group against those of Glyphinaphis and Cerataphis aphids with high bootstrap confidence levels, which strongly suggests that symbionts have been horizontally transferred from the aphids' lineage to the planthoppers'. Received: 29 March 2000 / Accepted: 31 May 2000     

Genes Involved in Cell Wall Localization and Side Chain Formation of Rhamnose-Glucose Polysaccharide in Streptococcus mutans

We identified in Streptococcus mutans six new genes (rgpA through rgpF), whose disruption results in a loss of serotype-specific antigenicity, specified by the glucose side chains of rhamnose-glucose polysaccharide from the cell wall. Rhamnose and glucose content of the cell wall decreased drastically in all these disruption mutants, except that in the rgpE mutant only the glucose content decreased. RgpC and RgpD are homologous to ATP-binding cassette transporter components and may be involved in polysaccharide export, whereas RgpE may be a transferase of side chain glucose.     

An automatic image analysis system for RLGS films

The RLGS (Restriction Landmark Genome Scanning) method was originally developed as a powerful method for enabling viewing of thousands of restriction landmarks. It offers a tool for obtaining information about genetic loci, with a single RLGS profile displaying approximately 2000 restriction landmarks as spots. One of the most useful applications is RLGS spot mapping, which allows the efficient, low-cost construction of the genetic map of any organism. However, analyses of the profiles depend mainly on human visual observation and are tedious and laborious. Although several commercially available image analyzing systems for profile comparison have been examined, they cannot be used for the RLGS spot mapping system owing to the background characteristics of the RLGS profiles, unsatisfactory rates of correspondence, and inefficient correction of informative genetic data. We therefore developed a novel automatic image analysis system for RLGS spot mapping, using an original algorithm based on the binary image transferred from the original RLGS profile. This system was employed for identifying non-polymorphic and parental strain-specific polymorphic spots of the F₁ mouse profile and yielded efficient initial screening of RLGS profiles. Received: 5 December 1997 / Accepted: 6 April 1998     

A New Metabolite,Parasiticolide A,from Aspergillus parasiticus

Two β-glucosidases, G1 and G2, were purified from the culture supernatant of Penicillium herquei Banier and Sartory. Both the purified enzymes were homogeneous on polyacrylamide disc gel electrophoresis. The molecular weights of G1 and G2 were estimated to be 125,000 and 122,000, respectively, by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. G1 and G2 contained 12.7% and 16.1% carbohydrate as glucose, and had isoelectric points of 5.02 and 5.24, respectively. Both enzymes had optimum pHs of 4.0~4.5 and optimum temperatures at 60°C, but pH - and thermo-stabilities of G1 were higher than those of G2. Both enzymes were active not only on p-nitrophenyl β-d-glucopyranoside, salicin, and the p-glucobioses tested but also on laminarin. CM-Cellulose was a very poor substrate for both enzymes. The activities of G1 toward the substrates except for laminarin and CM-cellulose were apparently higher than those of G2. Both enzymes acted on cellobiose to produce a transfer product.     

Nucleosome compaction facilitates HP1γ binding to methylated H3K9

The α, β and γ isoforms of mammalian heterochromatin protein 1 (HP1) selectively bind to methylated lysine 9 of histone H3 via their chromodomains. Although the phenotypes of HP1-knockout mice are distinct for each isoform, the molecular mechanisms underlying HP1 isoform-specific function remain elusive. In the present study, we found that in contrast to HP1α, HP1γ could not bind tri-methylated H3 lysine 9 in a reconstituted tetra-nucleosomes when the nucleosomes were in an uncompacted state. The hinge region connecting HP1''s chromodomain and chromoshadow domain contributed to the distinct recognition of the nucleosomes by HP1α and HP1γ. HP1γ, but not HP1α, was strongly enhanced in selective binding to tri-methylated lysine 9 in histone H3 by the addition of Mg²⁺ or linker histone H1, which are known to induce compaction of nucleosomes. We propose that this novel property of HP1γ recognition of lysine 9 in the histone H3 tail in different nucleosome structures plays a role in reading the histone code.     

Tubulin-polymerization inhibitors derived from thalidomide

2-(2,6-Diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione (5HPP-33), which was obtained during our previous structural development studies on thalidomide, was revealed to possess potent tubulin-polymerization-inhibiting activity, comparable to that of the known tubulin-polymerization inhibitors, rhizoxin and colchicine. A major metabolite of thalidomide, 5-hydroxythalidomide, which possesses a hydroxyl group at the position corresponding to that of 5HPP-33, also showed moderate inhibitory activity.