Effects of the phosphatase inhibitors,okadaic acid,ATPgammaS, and calyculin A on the dividing sand dollar egg

The effects of the phosphatase inhibitors, okadaic acid (OA), adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and calyculin A (CL-A) on anaphase chromosome movement, cytokinesis, and cytoskeletal structures at cell division were examined by being microinjected into mitotic sand dollar eggs. When OA was injected, chromosome movement was inhibited and, moreover, chromosomes were ejected from the polar regions of the mitotic apparatus. By immunofluorescence, microtubules were observed to be severed in the OA-injected eggs, causing the smooth cell surface to be changed to an irregular surface. When ATPgammaS and CL-A were injected, the effect on cell shape was remarkable: In dividing eggs, furrowing stopped within several seconds after injection, small blebs appeared on the cell surface and became large, spherical or dumbbell cell shapes then changed to irregular forms, and subsequently cytoplasmic flow occurred. Microfilament detection revealed that actin accumulation in the cortex, which was not limited to the furrow cortex, occurred shortly after injection. Cortical accumulation of actin is thought to induce force generation and random cortical contraction, and accordingly to result in bleb extrusion from the cortex. Consequently, the phosphatase inhibitors inhibited the transition from mitosis to interphase by mediating cortical accumulation of actin filaments and/or fragmentation of microtubules.     

MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.     

Single bacterial cell detection using a mutant luciferase

Previously, we constructed a genetically modified luciferase of Photinus pyralis that generates more than 10-fold higher luminescence intensity than the wild-type enzyme. In this study, we demonstrate that this modified luciferase enables us to detect ATP at 10⁻¹⁸ mol, which is almost equal to the quantity contained in a single bacterial cell. Consequently,we have been able to detect bacterial contamination in samples as low as one colony-forming unit (c.f.u.) for both Escherichia coli and Bacillus subtilis.     

Difference between Maturation Division and Cleavage in Starfish Oocytes: Dependency of Induced Cytokinesis on the Size of the Aster as Revealed by Transplantation of the Centrosome

Using the starfish oocyte and zygote, we investigated the abilities of the centrosome at maturation and cleavage divisions to form the aster and induce cytokinesis, in order to determine differences between these divisions. The transplanted centrosome originated from both maturation and cleavage, induced an additional furrow in cleavage in the recipient cells, but did not induce abnormal polar body formation at maturation. Although it organized an additional aster in the recipient cell in both divisions, a difference in size among asters formed was recognized. Therefore, mitotic asters were stabilized with hexylene glycol in order to measure their radius and clarify this difference. The mean radius (14.4 μm) of the first meiotic aster was significantly smaller than that (20.4 μm) of the aster at the first cleavage. The transplanted cleavage centrosome formed as small an aster as the recipient's own at maturation divisions. When zygotes were briefly treated with colcemid so that the zygotes could not perform cytokinesis but did perform karyokinesis, the size of aster became the same as that in meiosis. These results prove that although any centrosome functions as a microtubule organizing center independent of its origin, the size of the resultant aster decides whether or not cytokinesis would be induced.     

BAS1 and SOB7 act redundantly to modulate Arabidopsis photomorphogenesis via unique brassinosteroid inactivation mechanisms

Active brassinosteroids (BRs), such as brassinolide (BL) and castasterone (CS), are growth-promoting plant hormones. An Arabidopsis cytochrome P450 monooxygenase (CYP734A1, formerly CYP72B1), encoded by the BAS1 gene, inactivates BRs and modulates photomorphogenesis. BAS1 was identified as the overexpressed gene responsible for a dominant, BR-deficient mutant, bas1-D. This mutant was isolated in an activation-tagged screen designed to identify redundant genes that might not be identified in classic loss-of-function screens. Here we report the isolation of a second activation-tagged mutant with a BR-deficient phenotype. The mutant phenotype is caused by the overexpression of SOB7 (CYP72C1), a homolog of BAS1. We generated single and double null-mutants of BAS1 and SOB7 to test the hypothesis that these two genes act redundantly to modulate photomorphogenesis. BAS1 and SOB7 act redundantly with respect to light promotion of cotyledon expansion, repression of hypocotyl elongation and flowering time in addition to other phenotypes not regulated by light. We also provide biochemical evidence to suggest that BAS1 and SOB7 act redundantly to reduce the level of active BRs, but have unique mechanisms. Overexpression of SOB7 results in a dramatic reduction in endogenous CS levels, and although single null-mutants of BAS1 and SOB7 have the same level of CS as the wild type, the double null-mutant has twice the amount. Application of BL to overexpression lines of BAS1 or SOB7 results in enhanced metabolism of BL, though only BAS1 overexpression lines confer enhanced conversion to 26-OHBL, suggesting that SOB7 and BAS1 convert BL and CS into unique products.     

Sequence-specific and non-specific binding of the Rci protein to the asymmetric recombination sites of the R64 shufflon

Specific cleavages within the shufflon-specific recombination site of plasmid R64 were detected by primer extension when a DNA fragment carrying the recombination site was incubated with the shufflon-specific Rci recombinase. Rci-dependent cleavages occurred in the form of a 5' protruding 7 bp staggered cut, suggesting that DNA cleavage and rejoining in the shufflon system take place at these positions. As a result, shufflon crossover sites were designated as sfx sequences consisting of a central 7 bp spacer sequence, and left and right 12 bp arms. R64 sfx sequences are unique among various site-specific recombination sites, since only the spacer sequence and the right arm sequence are conserved among various R64 sfxs, whereas the left arm sequence is not conserved and is not related to the right arm sequence. From nuclease protection analyses, Rci protein was shown to bind to entire R64 and artificial sfx sequences, suggesting that one Rci molecule binds to the conserved sfx right arm in a sequence-specific manner and the second to the sfx left arm in a non-specific manner. The sfx left arm sequences as well as the right arm sequences were shown to determine affinity to Rci and subsequently inversion frequency. Asymmetry of the sfx sequence may be the reason why Rci protein acts only on the inverted sfx sequences.     

Crystallographic characterization of the crossed lamellar structure in the bivalve Meretrix lamarckii using electron beam techniques

To understand the formation mechanism of crossed lamellar structures in molluskan shells, the crystallographic structural features in the shell of a bivalve, Meretrix lamarckii, were investigated using scanning electron microscopy, electron backscattered diffraction, and transmission electron microscopy with a focused ion beam sample preparation technique. Approximately 0.5 μm-thick lamellae (the second-order units) are piled up obliquely toward the growth direction to form the first-order unit and the obliquity is inverted between adjacent units along the shell thickness direction. The first-order units originate around the center of the shell, initially growing parallel to the shell and subsequently curving toward the inner or outer surfaces. The lamellae consist of aragonite granular and columnar layers, which group together to adopt the same crystal orientation forming crystallographic units (crystallites). Multiple {1 1 0} twins are common both in the granular and columnar layers. The crystallite c-axis is parallel to the columns and is inclined at angles 0–50° from the lamellar normal (dispersing among individual lamellae), toward the shell growth direction. Probably, the directions of the a- and b-axes are random in the lamellae, showing no specific orientation.     

Identification of surrogate ligands for orphan G protein-coupled receptors

We prepared fusion proteins with an alpha subunit of G protein Gi (Gi1alpha) of 26 orphan G protein-coupled receptors (GPCRs) and with Gsalpha of 10 orphan GPCRs, most of which had been identified from the human genome previously [FEBS Lett 520 (2002) 97]. Ligands for these fusion proteins were screened from a library consisting of approximately 1000 authentic compounds by measuring their effect on [35S]GTPgammaS binding to membrane preparations of insect Sf9 cells expressing these fusion proteins. Eleven compounds were found to act as surrogate agonists for a GPCR-Gsalpha and four GPCR-Gialpha fusion proteins, a compound as an inverse agonist for two GPCR-Gsalpha fusion proteins, and a compound as an endogenous agonist for a GPCR-Gialpha fusion protein.     

Actinomycetal Community Structures in Seawater and Freshwater Examined by DGGE Analysis of 16S rRNA Gene Fragments

The actinomycetal community structures in marine and freshwater environments (the Pacific Ocean, East China Sea, Tokyo Bay, and Arakawa River) were investigated by a culture-independent molecular method to clarify spatial and seasonal distributions. Deoxyribonucleic acid (DNA) was extracted from environmental water samples, and a community analysis was carried out on polymerase chain reaction-amplified 16S ribosomal DNA. The amplified DNA fragments were analyzed by denaturing gradient gel electrophoresis (DGGE) and nonmetric multidimensional scaling analysis, followed by sequencing analysis. The actinomycetal community structures were different at each station in the Pacific Ocean, the East China Sea, Tokyo Bay, and Arakawa River, and different populations predominated in each area. There were vertical variations in actinomycetal communities in the Pacific Ocean and East China Sea between the surface and 100-m depth, but communities were similar from 200- to 1,000-m depths. There were also distinct seasonal variations in communities in Tokyo Bay. Phylogenetic analysis of DNA fragments recovered from DGGE bands revealed that most of the predominant actinomycetal strains were uncultured and were quite different from well known culturable strains, such as the Streptomyces, Micromonospora, Microbispora, Salinispora, and Actinoplanes groups. These results suggest that the marine environment is an attractive target for discovering new actinomycetal populations producing bioactive compounds and that sampling depth and season are important considerations for isolating various populations effectively.     

Diversity and functional analysis of proteorhodopsin in marine Flavobacteria

Proteorhodopsin (PR) genes are widely distributed among marine prokaryotes and functions as light-driven proton pump when expressed heterologously in Escherichia coli, suggesting that light energy passing through PR may be substantial in marine environment. However, there are no data on PR proton pump activities in native marine bacteria. Here, we demonstrate light-driven proton pump activity (c. 124 H(+) PR(-1) min(-1) ) in recently isolated marine Flavobacteria. Among 75 isolates, 38 possessed the PR gene. Illumination of cell suspensions from all eight tested strains in five genera triggered marked pH drops. The action spectrum of proton pump activity closely matched the spectral distribution of the sea surface green light field. Addition of hydroxylamine to a solubilized membrane fraction shifted the spectrum to a form characteristic of PR photobleached into retinal oxime, indicating that PRs in flavobacterial cell membranes transform the photon dose in incident radiation into energy in the form of membrane potential. Our results revealed that PR-mediated proton transport can create the sufficient membrane potential for the ATP synthesis in native flavobacterial cells.