Staggered movement of an actin filament sliding on myosin molecules in the presence of ATP

An actin filament sliding on myosin molecules in the presence of an extremely low concentration of ATP exhibited a staggered movement. Longitudinally sliding movement of the filament was frequently interrupted by its non-sliding, fluctuating movements both in the longitudinal and transversal directions. Intermittent sliding movements of an actin filament indicate establishment of a coordination of ATP-mediated active sites distributed along the filament.     

Glibenclamide blocks volume-sensitive Cl- channels by dual mechanisms

To study the mechanisms of glibenclamide actions onvolume-sensitive Clchannels, whole cell patch-clamp studies were performed at various pHlevels in human epithelial Intestine 407 cells. Extracellular application of glibenclamide reversibly suppressed volume-sensitive Cl currents in the entirerange of voltage examined (100 to +100 mV) and accelerated thedepolarization-induced inactivation at pH 7.5. When glibenclamide wasapplied from the intracellular side, in contrast, no effect wasobserved. At acidic pH, at which the weak acid glibenclamide existslargely in the uncharged form, the instantaneous current was, in avoltage-independent manner, suppressed by the extracellular drug atmicromolar concentrations without significantly affecting thedepolarization-induced inactivation. At alkaline pH, at which almostall of the drug is in the charged form, glibenclamide speeded theinactivation time course and induced a leftward shift of thesteady-state inactivation curve at much higher concentrations. Thus itis concluded that glibenclamide exerts inhibiting actions onswelling-activated Clchannels from the extracellular side and that the uncharged form ismainly responsible for voltage-independent inhibition of instantaneous currents, whereas the anionic form facilitates voltage-dependent channel inactivation in human epithelial Intestine 407 cells.

     

Genomic Analysis of the Viral Population in Genital Secretions Early after Infection of Simian Immunodeficiency Viruses in Macaque Monkeys

To clarify the change in the viral population during passage from the vaginal cavity to blood circulation and vice versa, we examined the viral clones detected in cells in vaginal washes (VWCs) early after inoculation and after systemic infection with polyclonal SIV. In two intravaginally inoculated monkeys, the viral clones found in VWCs at 18 days p.i. were shown to be some of those contained in the inoculum, whereas the viral population in the peripheral blood mononuclear cells (PBMCs) was a monotype. This gradual decrease of viral clones suggested the possible existence of two barriers, one at the genital tract and the other between the genital tract and the blood. Later, at one month p.i., the viral clones in VWCs became rather restricted, whereas those in PBMCs diverged from a single clone to several clones. This suggested that different mechanisms affect the viral populations in PBMCs and VWCs. In order to examine how the viral population was affected by passage from the blood to the vaginal cavity, a monkey was intravenously inoculated and the viral clones in VWCs were analyzed at 14 days p.i., at a time of the heterogeneous population in PBMCs. The viral population in VWCs was found to be a single clone and this clone was a minor type in PBMCs, suggesting that the major clone in PBMCs was not always secreted to the vaginal cavity.     

Genomic analysis of gamma-ray-induced germ-cell mutations at the b locus recovered from the medaka specific-locus test.

To study how gamma-ray-induced germ-cell mutations are fixed at the early embryonic stage of the next generation, genomic alterations in the b locus mutants (colorless melanophores) detected during development in the medaka specific-locus test (SLT) were analyzed. First, nine anonymous DNA markers linked to the b locus were cloned and mapped into the region extending about 47cM surrounding the b locus. Next, losses of paternal alleles of these DNA markers were examined in each of the 51 gamma-ray-induced b locus mutants obtained after irradiation of sperm or spermatids. In these mutants, 47 were dominant lethals, three were semi-viable and one was viable. All the mutants examined had large deletions surrounding the b locus. One viable mutant had an interstitial deletion, while all the semi-viable and dominant lethal ones appeared to have terminal deletions. Deletions extending about 20-35cM were the most frequently observed in 18 of the 51 mutants examined. The largest one extended more than 40cM. These results suggest that most of the gamma-ray induced germ cell mutations recovered as total specific-locus mutants were accompanied by large genomic deletions, which eventually led the mutant embryos to dominant lethality.     

Geographical variation in photoperiodic induction of larval diapause in the bruchid beetle, Bruchidius dorsalis: polymorphism in overwintering stages

The bruchid beetle, Bruchidius dorsalis Fahraeus (Coleoptera: Bruchidae), has a multivoltine life cycle and shows geographical variation of overwintering stages in Japan. Our previous study found that B. dorsalis enters larval diapause in the final instar under short photoperiods. In cooler areas, we observed that most individuals overwinter in the final larval stage in diapause, whereas beetles at different developmental stages (non‐diapausing young instars, diapausing instars, and adults) were overwintering in warmer areas. In this study, we investigated geographical variation in the photoperiodic response for induction of larval diapause at 20 °C (three populations) and 24 °C (two populations) to clarify the overwintering strategy of B. dorsalis. We observed that (1) diapause incidence at 20 °C changed sharply from ca. 100% to 0% with a change in photoperiod in all the populations, (2) critical photoperiod was longer at 20 °C in populations from cooler areas, and (3) critical photoperiod at 24 °C was shorter than at 20 °C and a fraction of the larvae did not enter diapause, even under short photoperiods. Overwintering stages estimated from these results were consistent with those actually observed in the field. This study indicates that the geographical variation of overwintering stages is likely to reflect adaptive diapause induction in each local environment.     

High-throughput production of recombinant antigens for mouse KIAA proteins in Escherichia coli: computational allocation of possible antigenic regions, and construction of expression plasmids of glutathione-S-transferase-fused antigens by an in vitro recombination-assisted method.

Since the end of 2001, we have conducted a project to isolate and determine entire sequences of mouse cDNA clones which encode the polypeptides corresponding to human KIAA proteins. Towards the ultimate goal of this project to clarify the biological functions of KIAA genes, we have set production of antibodies against mouse KIAA gene products based on their sequence information as the next important stage. As the first step, we developed a high-throughput system utilizing shotgun clones generated during entire sequencing of mouse KIAA cDNAs. The system consists of the following three parts: (1) Shotgun clones encoding regions suitable for production of antigens were selected using a newly developed browser system; (2) the protein-coding sequences of the selected shotgun clones were transferred into an expression vector by in vitro recombination-assisted method in a 96-well format, and expressed as glutathione S-transferase fusion proteins in Escherichia coli; and (3) the solubility of the recombinant antigens were preliminarily assessed in a small-scale culture and then large-scale production and purification was performed using glutathione-affinity beads or retrieval from polyacrylamide gels depending on their solubility. Using these systems, we successfully produced and purified 400 antigens for production of mKIAA antibodies to date.     

GALEIDINIIUM RUGATUM GEN. ET SP. NOV. (DINOPHYCEAE), A NEW COCCOID DINOFLAGELLATE WITH A DIATOM ENDOSYMBIONT1

A new sand‐dwelling dinoflagellate from Palau, Galeidinium rugatum Tamura et Horiguchi gen. et sp. nov., is described. The life cycle of this new alga consists of a dominant nonmotile phase and a brief motile phase. The motile cell transforms itself directly into the nonmotile cell after swimming for a short period, and cell division takes place in the nonmotile phase. The nonmotile cell possesses a dome‐like cell covering, which is wrinkled and equipped with a transverse groove on the surface. The cell has 10–20 chloroplasts and a distinct eyespot. The motile cell is Gymnodinium‐like in shape. The dinoflagellate possesses an endosymbiotic alga to which the chloroplasts belong and which is separated from the host (dinoflagellate) cytoplasm by a unit membrane. The endosymbiont cytoplasm also possesses its own eukaryotic nucleus and mitochondria. The eyespot is surrounded by triple membranes and is located in the host cytoplasm. Photosynthetic pigment analysis, using HPLC, revealed that G. rugatum possesses fucoxanthin as the principal accessory pigment instead of peridinin. The rbcL tree showed that G. rugatum is monophyletic with Durinskia baltica (Levander) Carty et Cox and Kryptoperidinium foliaceum (Stein) Lindemann and that this clade is closely related to the pennate diatom, Cylindrotheca sp. The endosymbiont of G. rugatum is therefore shown to be a diatom. Phylogenetic analysis based on small subunit rDNA sequences demonstrated that G. rugatum, D. baltica, and K. foliaceum, all of which are known to harbor an endosymbiont of diatom origin, are closely related.     

A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins II: public release of inaugural version of InGaP database containing gene/protein expression profiles for 127 mouse KIAA genes/proteins.

The inaugural version of the InGaP database (Integrative Gene and Protein expression database; http://www.kazusa.or.jp/ingap/index.html) is a comprehensive database of gene/protein expression profiles of 127 mKIAA genes/proteins related to hypothetical ones obtained in our ongoing cDNA project. Information about each gene/protein consists of cDNA microarray analysis, subcellular localization of the ectopically expressed gene, and experimental data using anti-mKIAA antibody such as Western blotting and immunohistochemical analyses. KIAA cDNAs and their mouse counterparts, mKIAA cDNAs, were mainly isolated from cDNA libraries derived from brain tissues, thus we expect our database to contribute to the field of neuroscience. In fact, cDNA microarray analysis revealed that nearly half of our gene collection is predominantly expressed in brain tissues. Immunohistochemical analysis of the mouse brain provides functional insight into the specific area and/or cell type of the brain. This database will be a resource for the neuroscience community by seamlessly integrating the genomic and proteomic information about the mouse KIAA genes/proteins.     

Characterization of a fission yeast mutant which displays defects in cell wall integrity and cytokinesis.

The fission yeast cps6-153 mutant was originally isolated based on its hypersensitivity to the spindle poison isopropyl N-3-chlorophenyl carbamate (CIPC). The mutant also shows defects in both cell wall integrity and cytokinesis, resulting in the accumulation of unseparated cells with weakened cell walls. The arrested cells display a disoriented alignment of cytoplasmic microtubules. When the mutant cells are cultivated at high temperature (35 degrees C), both cell walls and septa become very thick. Electron microscopy revealed the disorganized structure of the thickened cell walls and septa, in which fibrillar components were not completely masked with an amorphous matrix. rad25+ was cloned from a genomic library by complementation of the mutant phenotypes, suggesting the involvement of Rad25p, one of two 14-3-3 proteins in S. pombe, in the pathway of cell wall integrity and cytokinesis.