Genetic dissection of the formation of the forebrain in Medaka, Oryzias latipes

The forebrain, consisting of the telencephalon and diencephalon, is essential for processing sensory information. To genetically dissect formation of the forebrain in vertebrates, we carried out a systematic screen for mutations affecting morphogenesis of the forebrain in Medaka. Thirty-three mutations defining 25 genes affecting the morphological development of the forebrain were grouped into two classes. Class 1 mutants commonly showing a decrease in forebrain size, were further divided into subclasses 1A to 1D. Class 1A mutation (1 gene) caused an early defect evidenced by the lack of bf1 expression, Class 1B mutations (6 genes) patterning defects revealed by the aberrant expression of regional marker genes, Class 1C mutation (1 gene) a defect in a later stage, and Class 1D (3 genes) a midline defect analogous to the zebrafish one-eyed pinhead mutation. Class 2 mutations caused morphological abnormalities in the forebrain without considerably affecting its size, Class 2A mutations (6 genes) caused abnormalities in the development of the ventricle, Class 2B mutations (2 genes) severely affected the anterior commissure, and Class 2C (6 genes) mutations resulted in a unique forebrain morphology. Many of these mutants showed the compromised sonic hedgehog expression in the zona-limitans-intrathalamica (zli), arguing for the importance of this structure as a secondary signaling center. These mutants should provide important clues to the elucidation of the molecular mechanisms underlying forebrain development, and shed new light on phylogenically conserved and divergent functions in the developmental process.     

Analysis of Wnt8 for neural posteriorizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8

The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8 is the neural posteriorizing factor that acts upon neuroectoderm, we first assigned Frizzled 8c and Frizzled 9 to be functional receptors for Wnt8. We then, transplanted non-axial mesoderm into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induced the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of beta-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. We thus conclude that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis.     

Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1β-stimulated rat fibroblasts

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1β-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1β-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE₂, PGD₂, and PGF_2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1β-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A₂ (PLA₂) isozymes revealed that cytosolic PLA₂α, but not calcium-independent PLA₂s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1β stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1β stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.     

Early Staphylococcal Biofilm Formation on Solid Orthopaedic Implant Materials: In Vitro Study

Biofilms forming on the surface of biomaterials can cause intractable implant-related infections. Bacterial adherence and early biofilm formation are influenced by the type of biomaterial used and the physical characteristics of implant surface. In this in vitro research, we evaluated the ability of Staphylococcus epidermidis, the main pathogen in implant-related infections, to form biofilms on the surface of the solid orthopaedic biomaterials, oxidized zirconium-niobium alloy, cobalt-chromium-molybdenum alloy (Co-Cr-Mo), titanium alloy (Ti-6Al-4V), commercially pure titanium (cp-Ti) and stainless steel. A bacterial suspension of Staphylococcus epidermidis strain RP62A (ATCC35984) was added to the surface of specimens and incubated. The stained biofilms were imaged with a digital optical microscope and the biofilm coverage rate (BCR) was calculated. The total amount of biofilm was determined with the crystal violet assay and the number of viable cells in the biofilm was counted using the plate count method. The BCR of all the biomaterials rose in proportion to culture duration. After culturing for 2–4 hours, the BCR was similar for all materials. However, after culturing for 6 hours, the BCR for Co-Cr-Mo alloy was significantly lower than for Ti-6Al-4V, cp-Ti and stainless steel (P<0.05). The absorbance value determined in the crystal violet assay and the number of viable cells on Co-Cr-Mo were not significantly lower than for the other materials (P>0.05). These results suggest that surface properties, such as hydrophobicity or the low surface free energy of Co-Cr-Mo, may have some influence in inhibiting or delaying the two-dimensional expansion of biofilm on surfaces with a similar degree of smoothness.     

Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase

The antioncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell-cycle checkpoint regulation. Here we show that Chk2 associates with the oncogenic protein Wip1 (wild-type p53-inducible phosphatase 1) (PPM1D), a p53-inducible protein phosphatase. Phosphorylation of Chk2 at threonine68 (Thr68), a critical event for Chk2 activation, which is normally induced by DNA damage or overexpression of Chk2, is inhibited by expression of wild-type (WT), but not a phosphatase-deficient mutant (D314A) of Wip1 in cultured cells. Furthermore, an in vitro phosphatase assay revealed that Wip1 (WT), but not Wip1 (D314A), dephosphorylates Thr68 on phosphorylated Chk2 in vitro, resulting in the inhibition of Chk2 kinase activity toward glutathione S-transferase-Cdc25C. Moreover, inhibition of Wip1 expression by RNA interference results in abnormally sustained Thr68 phosphorylation of Chk2 and increased susceptibility of cells in response to DNA damage, indicating that Wip1 acts as a negative regulator of Chk2 in response to DNA damage.     

Interaction of N-ethyl-N'-nitro-n-nitrosoguanidine with nucleic acids and proteins in comparison with N-methyl-N'-nitro-N-nitrosoguanidine

Reactivity of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) was studied in comparison with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The radioactivity of [guanidino-14C]-ENNG was incorporated only into the protein fraction and that of [ethyl-14C]ENNG was incorporated into DNA, RNA and protein fractions in ascites hepatoma AH7974 cells, as were those of [guanidino-14C]- and [methyl-14C]MNNG, respectively. The amounts of the binding of ENNG were less than those of MNNG, especially in the corporation of the ethyl moiety of ENNG into nucleic acid fractions. In a non-cellular system, the radioactivity of [guanidino-14C]ENNG was incorporated into proteins, preferentially into basic proteins such as cytochrome c, but was not incorporated into nucleic acids. This behavior is similar to that of [guanidino-14C]MNNG, while the amount of binding of the former was about half of that of the latter. The radioactivity of [ethyl-14C]ENNG was also incorporated into basic proteins to almost the same extent as that of [methyl-14C]MNNG. However, the binding of the ethyl moiety of ENNG to nucleic acids was much lower than that of the methyl moiety of MNNG. Horse heart cytochrome c, bovine pancreatic RNase A and regenerating rat liver chromatin had altered their biological activities to various degrees after modification by ENNG or MNNG.     

Differences in the stemness characteristics and molecular markers of distinct human oral tissue neural crest‐derived multilineage cells

ObjectivesAlthough multilineage cells derived from oral tissues, especially the dental pulp, apical papilla, periodontal ligament, and oral mucosa, have neural crest‐derived stem cell (NCSC)‐like properties, the differences in the characteristics of these progenitor cell compartments remain unknown. The current study aimed to elucidate these differences.Material and methodsSphere‐forming apical papilla‐derived cells (APDCs), periodontal ligament‐derived cells (PDLDCs), and oral mucosa stroma‐derived cells (OMSDCs) from the same individuals were isolated from impacted developing teeth. All sphere‐forming cells were characterized through biological analyses of stem cells.ResultsAll sphere‐forming cells expressed neural crest‐related markers. The expression of certain tissue‐specific markers such as CD24 and CD56 (NCAM1) differed among tissue‐derived cells. Surprisingly, the expression of only CD24 and CD56 could be discriminated in human tissues. Although APDCs and PDLDCs exhibited greater mineralized cell differentiation than OMSDCs, they exhibited poorer differentiation into adipocytes in vitro. In immunocompromised mice, APDCs formed hard tissues better than PDLDCs and OMSDCs.ConclusionsAlthough cells with NCSC‐like properties present the same phenotype, they differ in the expression of certain markers and differentiation abilities. This study is the first to demonstrate the differences in the differentiation ability and molecular markers among multilineage human APDCs, PDLDCs, and OMSDCs obtained from the same patients, and to identify tissue‐specific markers that distinguish tissues in the developing stage of the human tooth with immature apex.

This study illustrates that neural crest‐derived cells from distinct oral tissues, namely the apical papilla, periodontal ligament, and oral mucosa, have varying differentiation potential, and tissue‐derived cell‐specific molecular markers have been identified. CD24 and NCAM1/CD56 expression were found to differ among multilineage oral tissue‐derived cells, similar to our observation in human tissue.     

Toward the Quantification of a Conceptual Framework for Movement Ecology Using Circular Statistical Modeling

To analyze an animal’s movement trajectory, a basic model is required that satisfies the following conditions: the model must have an ecological basis and the parameters used in the model must have ecological interpretations, a broad range of movement patterns can be explained by that model, and equations and probability distributions in the model should be mathematically tractable. Random walk models used in previous studies do not necessarily satisfy these requirements, partly because movement trajectories are often more oriented or tortuous than expected from the models. By improving the modeling for turning angles, this study aims to propose a basic movement model. On the basis of the recently developed circular auto-regressive model, we introduced a new movement model and extended its applicability to capture the asymmetric effects of external factors such as wind. The model was applied to GPS trajectories of a seabird (Calonectris leucomelas) to demonstrate its applicability to various movement patterns and to explain how the model parameters are ecologically interpreted under a general conceptual framework for movement ecology. Although it is based on a simple extension of a generalized linear model to circular variables, the proposed model enables us to evaluate the effects of external factors on movement separately from the animal’s internal state. For example, maximum likelihood estimates and model selection suggested that in one homing flight section, the seabird intended to fly toward the island, but misjudged its navigation and was driven off-course by strong winds, while in the subsequent flight section, the seabird reset the focal direction, navigated the flight under strong wind conditions, and succeeded in approaching the island.     

bfr1+, a novel gene of Schizosaccharomyces pombe which confers brefeldin A resistance, is structurally related to the ATP-binding cassette superfamily.

We have isolated a Schizosaccharomyces pombe gene, bfr1+, which on a multicopy plasmid vector, pDB248', confers resistance to brefeldin A (BFA), an inhibitor of intracellular protein transport. This gene encodes a novel protein of 1,531 amino acids with an intramolecular duplicated structure, each half containing a single ATP-binding consensus sequence and a set of six transmembrane sequences. This structural characteristic of bfr1+ protein resembles that of mammalian P-glycoprotein, which, by exporting a variety of anticancer drugs, has been shown to be responsible for multidrug resistance in tumor cells. Consistent with this is that S. pombe cells harboring bfr1+ on pDB248' are resistant to actinomycin D, cerulenin, and cytochalasin B, as well as to BFA. The relative positions of the ATP-binding sequences and the clusters of transmembrane sequences within the bfr1+ protein are, however, transposed in comparison with those in P-glycoprotein; the bfr1+ protein has N-terminal ATP-binding sequence followed by transmembrane segments in each half of the molecule. The bfr1+ protein exhibited significant homology in primary and secondary structures with two recently identified multidrug resistance gene products of Saccharomyces cerevisiae, Snq2 and Sts1/Pdr5/Ydr1. The bfr1+ gene is not essential for cell growth or mating, but a delta bfr1 mutant exhibited hypersensitivity to BFA. We propose that the bfr1+ protein is another member of the ATP-binding cassette superfamily and serves as an efflux pump of various antibiotics.     

A glutamine synthetase mutant of Saccharomyces cerevisiae shows defect in cell wall

To study the organization and biosynthesis of the yeast cell wall, hypo-osmolarity-sensitive mutants of Saccharomyces cerevisiae were analyzed. Cells of JS4 were irregular in shape and fragile. Calcofluor staining and quantitative analysis indicated that the chitin content was reduced. By DNA cloning and genetic analysis, the mutation hpo1-1 was found to be allelic to GLN1 which encodes glutamine synthetase. The glutamine content was significantly low in JS4, and the mutant was recovered from the cell wall defect by supplying glutamine in the medium. Partial inhibition of glutamine synthetase by phosphinothricin also induced defects in the cell wall. These results indicate that the shortage of glutamine affects cell wall integrity prior to other cellular functions.