Dissociation of the Nuf2-Ndc80 complex releases centromeres from the spindle-pole body during meiotic prophase in fission yeast

In the fission yeast Schizosaccharomyces pombe, centromeres remain clustered at the spindle-pole body (SPB) during mitotic interphase. In contrast, during meiotic prophase centromeres dissociate from the SPB. Here we examined the behavior of centromere proteins in living meiotic cells of S. pombe. We show that the Nuf2-Ndc80 complex proteins (Nuf2, Ndc80, Spc24, and Spc25) disappear from the centromere in meiotic prophase when the centromeres are separated from the SPB. The centromere protein Mis12 also dissociates during meiotic prophase; however, Mis6 remains throughout meiosis. When cells are induced to meiosis by inactivation of Pat1 kinase (a key negative regulator of meiosis), centromeres remain associated with the SPB during meiotic prophase. However, inactivation of Nuf2 by a mutation causes the release of centromeres from the SPB in pat1 mutant cells, suggesting that the Nuf2-Ndc80 complex connects centromeres to the SPB. We further found that removal of the Nuf2-Ndc80 complex from the centromere and centromere-SPB dissociation are caused by mating pheromone signaling. Because pat1 mutant cells also show aberrant chromosome segregation in the first meiotic division and this aberration is compensated by mating pheromone signaling, dissociation of the Nuf2-Ndc80 complex may be associated with remodeling of the kinetochore for meiotic chromosome segregation.     

Host-residual invariant NK T cells attenuate graft-versus-host immunity

Invariant NK T (iNKT) cells have an invariant TCR-alpha chain and are activated in a CD1d-restricted manner. They are thought to regulate immune responses and play important roles in autoimmunity, allergy, infection, and tumor immunity. They also appear to influence immunity after hemopoietic stem cell transplantation. In this study, we examined the role of iNKT cells in graft-vs-host disease (GVHD) and graft rejection in a mouse model of MHC-mismatched bone marrow transplantation, using materials including alpha-galactosylceramide, NKT cells expanded in vitro, and Jalpha18 knockout mice that lack iNKT cells. We found that host-residual iNKT cells constitute effector cells which play a crucial role in reducing the severity of GVHD, and that this reduction is associated with a delayed increase in serum Th2 cytokine levels. Interestingly, we also found that host-residual iNKT cause a delay in engraftment and, under certain conditions, graft rejection. These results indicate that host-residual iNKT cells attenuate graft-vs-host immunity rather than host-vs-graft immunity.     

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

Background

Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.

Results

We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages.

Conclusion

Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.     

Live cell imaging: approaches for studying protein dynamics in living cells

In the last decade, the long-standing biologist's dream of seeing the molecular events within the living cell came true. This technological achievement is largely due to the development of fluorescence microscopy technologies and the advent of green fluorescent protein as a fluorescent probe. Such imaging technologies allowed us to determine the subcellular localization, mobility and transport pathways of specific proteins and even visualize protein-protein interactions of single molecules in living cells. Direct observation of such molecular dynamics can provide important information about cellular events that cannot be obtained by other methods. Thus, imaging of protein dynamics in living cells becomes an important tool for cell biology to study molecular and cellular functions. In this special issue of review articles, we review various imaging technologies of microscope hardware and fluorescent probes useful for cell biologists, with a focus on recent development of live cell imaging.     

Effects of nutrient loadings from catchments on Asajino mire, a small coastal ombrotrophic mire in northernmost Japan

The relationship between water chemistry and vegetation was studied in a coastal ombrotrophic mire in northern Hokkaido, Japan. The distributions of Sphagnum and Phragmites communities were separated clearly by the pH and ion concentration of the peat surface-pore water. The drainage ditches along the road across the center of the mire had a high pH and ion concentration, as did the peat water in the western part of the mire. It was found that fields used for livestock farming on a hill to the west of the mire leached materials into the mire through drainage ditches, surface runoff, and probably also through ground water, and thus influenced the water chemistry of the mire. Management of the water, including that in the catchment of the mire, should be introduced before biological buffering capacity against excess nutrient loading caused by human activity is exceeded and the mire loses its ombrotrophic status.     

Synthesis of 6-Methyluridine via Palladium-Catalyzed Cross-Coupling Between A 6-Iodouridine Derivative and Tetramethylstannane

Abstract

6-Methyluridine can be synthesized from 5′-O-(tert-butyl-dimethylsilyl)-6-iodo-2′,3′-O-isopropylideneuridine via palladiumcatalyzed cross-coupling with Me₄Sn followed by deprotection. Application of this method for the synthesis of 6-phenyluridine was also carried out.     

Quantitative analyses of morphological changes of cell growth by graphics computer techniques. A methodologic study

A computerized image analysis system is described for quantitation of changes in cell morphology for use in the bioassay of certain types of growth factors in organ cultures of connective tissue cells.Eight response variables, indicative of cell growth, were measured (area of cell, area of nucleus, area of cytoplasm, length of the cell, lengths of the major and minor axes, number of nucleoli per cell, and total area of nucleoli per cell).Principal components analyses were used to eliminate redundant variables and to estimate the dimensionality of the problem. In general, only four response variables were required in order to describe the morphological changes occurring during cell growth of these connective tissue fibroblasts. Some sacrifice in independence of variables was tolerated in order to compensate for some shifts in correlations between some variables at different doses of growth factor. Cell length and area of the cell provided the best quantitative measures for distinguishing between doses of growth factors and for correct ranking of doses.     

Synthesis and Biological Evaluation of 1′-C-Cyano-Pyrimidine Nucleosides

Abstract

2′-Deoxy-, 2′-bromo-, and arabino-1′-C-cyano-pyrimidine nucleosides were synthesized from O² ,2′-cyclouridine. Incorporation of cyano group at the anomeric position was achieved by treatment of 1′,2′-unsaturated uridine with NBS in the presence of pivalic acid followed by TMS-cyanide and stannic chloride. Antineoplastic and antiviral activities of those compounds are also discussed.

     

Fractionated irradiation improves the mating performance of the West Indian sweet potato weevil Euscepes postfasciatus

• 1 The sterile insect technique (SIT) is widely used to suppress or eradicate target pest insect populations.
• 2 The effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females. The use of gamma radiation to induce sterility, however, negatively affects both somatic cells as well as reproductive cells. Consequently, mating performance of sterilized individuals decreases drastically over time. The mating propensity of sterilized Euscepes postfasciatus (Fairmaire) males irradiated with a single dose of 150 Gy (the current standard of the Okinawa Prefecture SIT programme) is equal to that of non‐irradiated weevils for the first 6 days.
• 3 Fractionated irradiation, in which a sterilizing dose is delivered over time in a series of smaller irradiations, reduces the damage of irradiation in insects. In the present study, we evaluated the effect of fractionated irradiation on male fertilization ability, longevity and mating propensity of E. postfasciatus for a period of 16 days after irradiation.
• 4 Although fractionated irradiation totalling 150 Gy was found to induce full sterility regardless of the number of individual doses, the mating propensity of male weevils sterilized by fractionated irradiation was maintained for the first 12 days. These results demonstrate that fractionated irradiation can be highly advantageous in programmes aimed at eradication of E. postfasciatus.