Inactivation of p34cdc2 kinase by the accumulation of its phosphorylated forms in porcine oocytes matured and aged in vitro.

Culturing of matured porcine oocytes in vitro results in the enhancement of their cytoplasmic ability for oocyte activation (so-called ageing), although they are arrested at metaphase II. The enhanced ability for oocyte activation is related to decreased activity of the maturation promoting factor (MPF). In the present study we clarified the molecular mechanism of MPF inactivation during ageing, especially the changes in the phosphorylation status of p34cdc2, a catalytic subunit of MPF, compared with that in fertilised oocytes. The MPF activity decreased gradually when maturation culture was prolonged from 36 to 72 h, confirming the decreasing MPF activity in aged oocytes. The activity of 48 h matured oocytes also decreased after in vitro fertilisation. Immunoblotting of p34cdc2 with anti-PSTAIRE antibody revealed that the culturing of matured oocytes induces a gradual increase in pre-MPF, which is a p34cdc2 and cyclin B complex inactivated by phosphorylation at the inhibitory phosphorylation site of p34cdc2. In contrast, pre-MPF decreased after fertilisation, indicating the degradation of cyclin B. These results suggest that the molecular mechanisms of inactivation of MPF are different between oocyte activation and ageing, and that the mechanism during ageing might be based on the inhibitory phosphorylation of p34cdc2, whereas that of oocyte activation is based on the degradation of cyclin B.     

Small molecular weight GTP-binding proteins in human erythrocyte ghosts

GTP-binding proteins (G proteins) were extracted from human erythrocyte ghosts by sodium cholate and purified by gel filtration on an Ultrogel AcA-44 column followed by hydroxyapatite column chromatography. At least two peaks of G proteins were separated by hydroxyapatite column chromatography. The second peak contained G proteins recognized by the antibodies against the respective alpha subunits of Gs and Gi, and the ras protein, while the G protein of the first peak was not recognized by any of these antibodies. The G protein of the first peak was purified further by Mono Q HR5/5 column chromatography. The purified G protein showed a molecular weight of about 22 kDa on SDS-polyacrylamide gel electrophoresis. This G protein (22K G) specifically bound guanosine 5'-(3-O-thio) triphosphate (GTP gamma S), GTP and GDP with a Kd value for GTP gamma S of about 50 nM. GTP gamma S-binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. The G proteins recognized by the antibodies against the alpha subunit of Go and the ADP-ribosylation factor for Gs, designated as ARF, were not detected in human erythrocyte ghosts. These results indicate that there are at least two species of small molecular weight G proteins in human erythrocyte ghosts: one is the ras protein and the other is a novel G protein of 22K G.     

Separate analysis of nuclear and cytosolic Ca2+ concentrations in human umbilical vein endothelial cells

Ca²⁺ concentration inside human umbilical vein endothelial cells was studied separately in cytosol and nucleus by a confocal laser scanning microscopy using fluo-3. The in vivo calibration curve for cytosol and nucleus showed good linearity between fluorescence intensity and Ca²⁺ concentration in cytosol ([Ca²⁺]_i) and nuclei ([Ca²⁺]_n). After calibration, [Ca²⁺]_n was constantly higher than [Ca²⁺]_i before and after the chelation of extracellular Ca²⁺ suggesting an active Ca²⁺ accumulation system on nuclear membrane. [Ca²⁺]_n was also constantly higher than [Ca²⁺]_i after the stimulation of thrombin (0.05 U/ml), FCS (10%), and thapsigargin (Tsg, 1μM). The temporal change of [Ca²⁺]_n and [Ca²⁺]_i was identical, and [Ca²⁺]_i gradient towards the nucleus and peripheral or central [Ca²⁺]_n rise was observed after these stimulations. From these results, [Ca²⁺]_n is not only regulated by the active Ca²⁺ accumulation system on nuclear membrane at rest but also the generation of Inositol-triphosphate. FCS caused heterogeneous [Ca²⁺]_n or [Ca²⁺]_i rise from cell to cell; single spike or oscillatory change of [Ca²⁺]_n and [Ca²⁺]_i was observed in about 56% of cells, which were completely abolished by the chelation of extracellular Ca²⁺, suggesting that FCS stimulated [Ca²⁺]_n and [Ca²⁺]_i rise solely depending on Ca²⁺ influx from extracellular medium. The higher concentration of [Ca²⁺]_n and heterogeneous [Ca²⁺]_n rise may have important roles in nuclear-specific cellular responses. © 1996 Wiley-Liss, Inc.     

Structural requirements of dictyopyrones isolated from Dictyostelium spp. in the regulation of Dictyostelium development and in anti-leukemic activity

Cellular slime molds are fascinating to the field of developmental biology, and have long been used as excellent model organisms for the study of various aspects of multicellular development. We have recently isolated alpha-pyronoids, named dictyopyrones A-D (1-4), from various species of Dictyostelium cellular slime molds, and it was shown that compound 3 may regulate Dictyostelium development. In this study, we synthesized dictyopyrones A-D (1-4) and their analogues, investigated the physiological role of the molecules in cell growth and morphogenesis in D. discoideum, and further verified their effects on human leukemia K562 cells. Nitrogen-containing compounds 22 and 37 strongly inhibited cell growth in K562 leukemia cells, indicating that these compounds may be utilized as novel lead compounds for anti-leukemic agents.     

A novel method for biomaterial scaffold internal architecture design to match bone elastic properties with desired porosity

An often-proposed tissue engineering design hypothesis is that the scaffold should provide a biomimetic mechanical environment for initial function and appropriate remodeling of regenerating tissue while concurrently providing sufficient porosity for cell migration and cell/gene delivery. To provide a systematic study of this hypothesis, the ability to precisely design and manufacture biomaterial scaffolds is needed. Traditional methods for scaffold design and fabrication cannot provide the control over scaffold architecture design to achieve specified properties within fixed limits on porosity. The purpose of this paper was to develop a general design optimization scheme for 3D internal scaffold architecture to match desired elastic properties and porosity simultaneously, by introducing the homogenization-based topology optimization algorithm (also known as general layout optimization). With an initial target for bone tissue engineering, we demonstrate that the method can produce highly porous structures that match human trabecular bone anisotropic stiffness using accepted biomaterials. In addition, we show that anisotropic bone stiffness may be matched with scaffolds of widely different porosity. Finally, we also demonstrate that prototypes of the designed structures can be fabricated using solid free-form fabrication (SFF) techniques.     

Membrane-associated IL-1 contributes to chronic synovitis and cartilage destruction in human IL-1 alpha transgenic mice

IL-1 molecules are encoded by two distinct genes, IL-1alpha and IL-1beta. Both isoforms possess essentially identical activities and potencies, whereas IL-1alpha, in contrast to IL-1beta, is known to act as a membrane-associated IL-1 (MA-IL-1) and plays an important role in a variety of inflammatory situations. The transgenic (Tg) mouse line (Tg1706), which was generated in our laboratory, overexpresses human IL-1alpha (hIL-1alpha) and exhibits a severe arthritic phenotype characterized by autonomous synovial proliferation with subsequent cartilage destruction. Because the transgene encoded Lys(64) to Ala(271) of the hIL-1alpha amino acid sequence, Tg mice may overproduce MA-IL-1 as well as soluble IL-1alpha. The present study investigated whether MA-IL-1 contributes to synovial proliferation and cartilage destruction in the development of arthritis. Flow cytometric analysis revealed that both macrophage-like and fibroblast-like synoviocytes constitutively produce MA-IL-1. D10 cell proliferation assay revealed MA-IL-1 bioactivity of paraformaldehyde-fixed synoviocytes and the further induction of endogenous mouse MA-IL-1 via autocrine mechanisms. MA-IL-1 expressed on synoviocytes triggered synoviocyte self-proliferation through cell-to-cell (i.e., juxtacrine) interactions and also promoted proteoglycan release from the cartilage matrix in chondrocyte monolayer culture. Interestingly, the severity of arthritis was significantly correlated with MA-IL-1 activity rather than with soluble IL-1alpha activity or concentration of serum hIL-1alpha. Moreover, when the Tg1706 line was compared with the Tg101 line, which selectively overexpresses the 17-kDa mature hIL-1alpha, the severity of arthritis was significantly higher in the Tg1706 line than in the Tg101 line. These results suggest that MA-IL-1 contributes to synoviocyte self-proliferation and subsequent cartilage destruction in inflammatory joint disease such as rheumatoid arthritis.     

Secretory production of<Emphasis Type="Italic"> Aspergillus oryzae</Emphasis> xylanase XynF1,<Emphasis Type="Italic"> xynF1</Emphasis> cDNA product,in the basidiomycete<Emphasis Type="Italic"> Coprinus cinereus</Emphasis>

The signal peptide of Aspergillus oryzae endo-(1,4)--xylanase XynF1 contains a C-terminal serine-arginine that directs efficient secretion of the enzyme into the culture medium. In the basidiomycete Coprinus cinereus, however, there is little secretion of XynF1 into the culture medium. Modification of the C-terminal sequence of the signal peptide to lysine-arginine resulted in efficient secretion of C. cinereus XynF1, suggesting the presence of a KEX2-like protease in this fungus.     

gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group

Bacillus anthracis, the causative agent of the human disease anthrax, Bacillus cereus, a food-borne pathogen capable of causing human illness, and Bacillus thuringiensis, a well-characterized insecticidal toxin producer, all cluster together within a very tight clade (B. cereus group) phylogenetically and are indistinguishable from one another via 16S rDNA sequence analysis. As new pathogens are continually emerging, it is imperative to devise a system capable of rapidly and accurately differentiating closely related, yet phenotypically distinct species. Although the gyrB gene has proven useful in discriminating closely related species, its sequence analysis has not yet been validated by DNA:DNA hybridization, the taxonomically accepted "gold standard". We phylogenetically characterized the gyrB sequences of various species and serotypes encompassed in the "B. cereus group," including lab strains and environmental isolates. Results were compared to those obtained from analyses of phenotypic characteristics, 16S rDNA sequence, DNA:DNA hybridization, and virulence factors. The gyrB gene proved more highly differential than 16S, while, at the same time, as analytical as costly and laborious DNA:DNA hybridization techniques in differentiating species within the B. cereus group.