Rapid patterning of Dictyostelium discoideum cells under confined geometry and its relation to differentiation

The following was recently reported by Bonner et al. (1995): (1) Rapid differentiation occurred into two zones in Dictyostelium discoideum cells confined in a fine glass capillary. The cells in the anterior zone exposed to the air appear similar to prestalk cells, while the posterior zone isolated from the air mimics prespore cells. (2) The volumes of the two zones are proportional to each other for different sized cell masses, and the proportion is the same as that in normal migrating slugs. We investigated the nature of this newly discovered rapid differentiation in a slightly modified geometry. Exponentially growing cells were harvested, washed to remove external nutrients, and pelleted by centrifugation. Subsequently, a small drop of the pelleted (starved) cells was placed on a slide glass and then confined in a two-dimensional space between the slide glass and a coverslip, with help of spacers whose thickness varied from 25 to 100 μm. As a result, a dark zone, which looked optically different, emerged within several minutes in the periphery of the disc of the confined cells, corresponding to the zonation in a capillary as previously reported. When the width of the peripheral zone was measured for more than 30 samples of different diameters for each thickness of the spacers, the width was found to be always about 100 μm, irrespective of the size difference of the cell mass placed. This seems to be contradictory to the previous observation made by Bonner et al. (1995). We also examined oxygen concentration dependence on the zone width. The zone width was found to be independent of the oxygen concentration at low concentrations, but increased rapidly at high concentrations. A reaction-diffusion mechanism for formation of the zone and possible involvement of atmospheric oxygen (O₂) in the initial steps of cell differentiation and pattern formation is discussed.     

Cyclic AMP induces phosphorylation of claudin-5 immunoprecipitates and expression of claudin-5 gene in blood-brain-barrier endothelial cells via protein kinase A-dependent and -independent pathways

Cyclic AMP (cAMP) promotes functions of tight junctions in endothelial cells, although its target remains unknown. We showed here that cAMP increased gene expression of claudin-5 and decreased that of claudin-1 in porcine blood-brain-barrier endothelial cells via protein kinase A (PKA)-independent and -dependent pathways, respectively. cAMP also enhanced immunoreactivity of claudin-5 along cell borders and in the cytoplasm, reorganized actin filaments, and altered signals of claudin-5, occludin, ZO-1, and ZO-2 along cell boundaries from zipperlike to linear patterns. In contrast, claudin-1 was detected only in the cytoplasm in a dotlike pattern, and its immunolabeling was reduced by cAMP. Interestingly, 31- and 62-kDa claudin-5 immunoprecipitates in the NP-40-soluble and -insoluble fractions, respectively, were highly phosphorylated on threonine residue(s) upon cAMP treatment. All these changes induced by cAMP, except for claudin-5 expression and its signals in the cytoplasm, were reversed by an inhibitor of PKA, H-89. We also demonstrated that cAMP elevated the barrier function of tight junctions in porcine blood-brain-barrier endothelial cells in PKA-dependent and -independent manners. These findings indicate that both PKA-induced phosphorylation of claudin-5 immunoprecipitates and cAMP-dependent but PKA-independent induction of claudin-5 expression could be involved in promotion of tight-junction function in endothelial cells.     

Tremorgenic Mycotoxin from Penicillium paraherquei

A tremorgenic mycotoxin was isolated from Penicillium paraherquei Abe ex G. Smith and identified as verruculogen. It was produced at the rate of approximately 1 mg/g of the dried fungal mycelium cultured on peptone-enriched Czapek-Dox medium at 28°C.     

Purification and characterization of a purple acid phosphatase from rat spleen

An acid phosphatase species which is activated by Fe2+ was purified 3,700-fold from rat spleen by chromatography on columns containing Blue-Sepharose, concanavalin A-Sepharose, Sephadex G-100, and CM-Sephadex. The enzyme hydrolyzed aryl phosphates, nucleoside di- and triphosphates, phosphoproteins, and thiamine pyrophosphate with Km values of 10(-4) to 10(-3) M at an optimal pH of 5.0-5.8. Co-purification of the acid phosphatase and acid phosphoprotein phosphatase indicated that they were identical. The purified enzyme was glycoprotein in nature, showing four heterogeneous forms on acid polyacrylamide gel electrophoresis (pI values, 7.8, 8.0, 8.3, and 8.5), but it gave a molecular weight of 33,000 on sodium dodecyl sulfate-gel electrophoresis and gel permeation chromatography. The enzyme had a purple color (lambda max 545 nm) and contained 2 iron atoms per enzyme molecule. Among reductants, ascorbic acid and Fe2+ were the best activators, although their combined effect was not additive. Fe2+ and ascorbic acid both changed the purple enzyme into the same active form (lambda max 515 nm), giving almost the same kinetic constants for substrates and for inhibitors such as molybdate, phosphate and fluoride. However, low concentrations of Fe2+, from 0.01 mM to 1.0 mM, immediately and reversibly activated the enzyme, whereas high concentrations of ascorbic acid over 1 mM were required for maximal activation, which was slow and irreversible.     

The Hsp90 Inhibitor Geldanamycin Abrogates Colocalization of eIF4E and eIF4E-Transporter into Stress Granules and Association of eIF4E with eIF4G

The eukaryotic translation initiation factor eIF4E plays a critical role in the control of translation initiation through binding to the mRNA 5′ cap structure. eIF4E is also a component of processing bodies and stress granules, which are two types of cytoplasmic RNA granule in which translationally inactivated mRNAs accumulate. We found that treatment with the Hsp90 inhibitor geldanamycin leads to a substantial reduction in the number of HeLa cells that contain processing bodies. In contrast, stress granules are not disrupted but seem to be only partially affected by the inhibition of Hsp90. However, it is striking that eIF4E as well as its binding partner eIF4E transporter (4E-T), which mediates the import of eIF4E into the nucleus, are obviously lost from stress granules. Furthermore, the amount of eIF4G that is associated with the cap via eIF4E is reduced by geldanamycin treatment. Thus, the chaperone activity of Hsp90 probably contributes to the correct localization of eIF4E and 4E-T to stress granules and also to the interaction between eIF4E and eIF4G, both of which may be needed for eIF4E to acquire the physiological functionality that underlies the mechanism of translation initiation.     

Development of a novel fusogenic viral liposome system (HVJ-liposomes) and its applications to the treatment of acquired diseases

Toward human gene therapy and gene analysis in vivo, a novel hybrid vector based on liposome has been developed for more efficient gene delivery and gene expression. The liposome was decorated with HVJ (Sendai virus) envelope fusion proteins to introduce DNA directly into the cytoplasm, and contained DNA and DNA-binding nucelar protein to enhance expression of the gene. Recently, several types of HVJ-liposomes were developed by altering the lipid components of the liposomes. HVJ-cationic liposomes increased gene delivery 100 - 800 times more efficiently in vitro than the conventional HVJ-anionic liposomes. HVJ-cationic liposomes were also more useful for gene expression in restricted portions of organs and for gene therapy of disseminated cancers. It was further discovered that the use of anionic liposomes with a virus-mimicking lipid composition (HVJ-AVE liposomes) increased transfection efficiency by several fold in vivo, especially in liver and muscle. By coupling the Epstein-Barr (EB) virus replicon apparatus to HVJ-liposomes, transgene expression was sustained in vitro and in vivo. Most animal organs were found to be suitable targets for the fusigenicviral liposome system, and numerous gene therapy strategies using this system were successful in animals.     

Responses of coral reefs to increased amplitude of sea-level changes at the Mid-Pleistocene Climate Transition

We show responses of coral reefs to increased amplitude of sea-level changes at the Mid-Pleistocene Climate Transition (MPT) based on lithostratigraphic, sedimentologic and calcareous nannofossil biostratigraphic investigations on Pleistocene reef-complex deposits (Ryukyu Group) on the Motobu Peninsula, Okinawa-jima, Central Ryukyus. Our data show that reef growth started in earliest Quaternary time (1.45-1.65 Ma) and that extensive reef formation dates back to ∼ 0.8 Ma. The mode of Quaternary sedimentation changed at ∼ 0.8 Ma in the study area. Before this time, thick siliciclastics and mixed carbonate-siliciclastics accumulated, which were followed by the deposition of bioclastic sediments (detrital limestone). No indications have been found of episodic subaerial exposures in these deposits and no calcareous nannofossil biozones are lacking. Since the detrital limestone includes biogenic components characterizing fore-reef to shelf environments, the coastal areas of the northern Motobu Peninsula mostly lay in fore-reef to shelf environments for > 0.6 million years (between ∼ 0.8 Ma and 1.45-1.65 Ma), when the sediments had not been subaerially exposed due to sea-level changes characterized by relatively small amplitudes. Coral limestone that formed in the latest Early to Middle Pleistocene between 0.4 Ma and 0.8 Ma extends over the study area, ranging in elevation from 0 to 70 m. This coral limestone grades upward into fore-reef to shelf carbonates (rhodolith, Cycloclypeus-Operculina, and detrital limestones) which is in turn overlain by coral limestone. This succession, combined with configuration of the lithofacies and paleobathymetry inferred from lithology and biogenic components, implies that the reef-complex deposits formed responding to sea-level changes with amplitude of > 60 m. Consequently, we suggest that the change in the mode of sedimentation results from increased amplitude of sea-level fluctuations at ∼ 0.8 Ma. This timing corresponds roughly to the timing of the Mid-Pleistocene Climate Transition (MPT).