Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.     

Roles of Poly(3-Hydroxybutyrate) Depolymerase and 3HB-Oligomer Hydrolase in Bacterial PHB Metabolism

Many poly-3-hydroxybutyrate (PHB)-degrading enzymes have been studied. But biological roles of 3HB-oligomer hydrolases (3HBOHs) and how PHB depolymerases (PHBDPs) and 3HBOHs cooperate in PHB metabolism are not fully elucidated. In this study, several PHBDPs and 3HBOHs from three types of bacteria were purified, and their substrate specificity, kinetic properties, and degradation products were investigated. From the results, PHBDP and 3HBOH seemed to play a role in PHB metabolism in three types of bacteria, as follows: (A) In Ralstonia pickettii T1, an extracellular PHBDP degrades extracellular PHB to various-sized 3HB-oligomers, which an extracellular 3HBOH hydrolyzes to 3HB-monomers. (B) In Acidovorax sp. SA1, an extracellular PHBDP hydrolyzes extracellular PHB to small 3HB-oligomers (dimer and trimer), which an intracellular 3HBOH efficiently degrades to 3HB in the cell. (C) In Ralstonia eutropha H16, an intracellular 3HBOH helps in the degradation of intracellular PHB inclusions by PHBDP.     

Multiple sex chromosome system of X1X1X2X2/X1X2)Y type in lutjanid fish, Lutjanus quinquelineatus (Perciformes)

The karyotype and other chromosomal markers as revealed by C-banding and Ag-staining were studied in Lutjanus quinquelineatus and L. kasmira (Lutjanidae, Perciformes). While in latter species, the karyotype was invariably composed of 48 acrocentric chromosomes in both sexes, in L. quinquelineatus the female karyotype had exclusively 48 acrocentric chromosomes (2n = 48) but that of the male consisted of one large metacentric and 46 acrocentric chromosomes (2n = 47). The chromosomes in the first meiotic division in males showed 22 bivalents and one trivalent, which was formed by an end-to-end association and a chiasmatic association. Multiple sex chromosome system of X₁X₁X₂X₂/X₁X₂Y type resulting from single Robertsonian fusion between the original Y chromosome and an autosome was hypothesized to produce neo-Y sex chromosome. The multiple sex chromosome system of L. quinquelineatus appears to be at the early stage of the differentiation. The positive C-banded heterochromatin was situated exclusively in centromeric regions of all chromosomes in both species. Similarly, nucleolus organizer region sites were identified in the pericentromeric region of one middle-sized pair of chromosomes in both species. The cellular DNA contents were the same (3.3 pg) between the sexes and among this species and related species.     

Relationship Between Curved DNA Conformations and Slow Gel Migration

Abstract

We propose some specific DNA conformations that explain, in terms of molecular conformations, the anomalous gel electrophoretic behavior of the sequences (VA₄T₄X)₁, and (V₂A₃X₂)₁ where V and X are either G or C. Previously (J. Biomole. Struct. Dyn. 4, 41, 1986) we considered hydrophobic interactions a mong aliphatic hydrocarbon groups in A/T sequences. In the sequences (T)_n · (A)_n, the T's are slightly bent to yield structures with tightly stacked methyl groups along one side of the major groove. By folding together the two pairs of stacked methyls on the opposite sides of the major groove, TTAA might yield a relatively sharp bend. On this basis, we show below that the sequences (VT₄A₄X)₁ might form a very tightly coiled super-helix whereas the sequences (VA₄T₄X)₁ form a broad super-helix of radius ~ 120 A for i = 25. The sequence (V₂A₃T₃X₂)₁ forms a slightly smaller radius super-helix. The time of passage through the gel has been taken to be inversely proportional to the smallesuiimension of the molecule. Specifically we are taking the ratio of the apparent molecular weight to the actual molecular weight to be related to the moment of inertia I₁ about the smallest principal axis of the molecular conformation. We find a good fit to the experimental gel mobility data of Hagerman (2) if we assume this ratio to be proportional to (I₁)^1/5.     

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.     

Isolation and Biological Activity of a Peptidal Antibiotic P168

The structure of the xyloglucan synthesised in vitro by the particulate fraction of suspension-cultured soybean (Glycine max) cells from UDP-glucose and UDP-xylose is mainly composed of two kinds of oligosaccharide-building blocks, a heptasaccharide unit and a pentassaccharide unit [T. Hayashi and K. Matsuda, J. Biol Chem., 256, 11117 (1981)]. The synthesis of the pentasaccharide unit is probably the first step in the construction of oligosaccharide building blocks to elongate the ²-1,4-glucan backbone. This enzymatically synthesized xyloglucan was shown to have the same molecular size (M_w, 180,000) as the xyloglucan prepared from soybean cell walls by gel filtration on a Sepharose CL-6B column, and the same building blocks distributed among each fraction. A pulse-chase experiment indicated that the pentasaccharide unit was converted into the heptasaccharide unit. The conversion was regulated by the concentration of UDP-xylose.     

Three-dimensional common-feature hypotheses for octopamine agonist 2-(arylimino)imidazolidines.

Three-dimensional pharmacophore hypotheses were built from a set of 10 octopamine (OA) agonist 2-(Arylimino)imidazolidines (AIIs), 2-(Arylimino)thiazolidines (AITs) and 2-(Arylimino)oxazolidines (AIOs). Among the 10 common-featured models generated by program Catalyst/HipHop, a hypothesis including a ring aromatic (RA), a positive ionizable (PI) and three hydrophobic aliphatic (HpAl) features was considered to be important in evaluating the OA-agonist activity. Active OA agonist 2,6-Et2 AII mapped well onto all the RA, PI and HpAl features of the hypothesis. On the other hand, less active compounds were shown to be difficult to achieve the energetically favorable conformation which is found in the active molecules in order to fit the 3-D common-feature pharmacophore models. Taken together, 2,6-Et2-Ph and foramidine structures are important as OA agonists. The present studies on OA agonists demonstrate that a RA, a PI and three HpAl sites located on the molecule seem to be essential for OA-agonist activity.     

Potent effects of,and mechanisms for,modification of crosstalk between macrophages and adipocytes by lactobacilli

The murine macrophage‐like cell line J774.1 was treated with heat‐killed cells of Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC 0356). Interleukin (IL)‐6, IL‐12, and tumor necrosis factor‐α were profiled from the J774.1 cells using enzyme‐linked immunosorbent assay methods. The conditioned medium from cultured J774.1 cells was transferred to the preadipocyte cell line 3T3‐L1 (which is a mouse embryonic fibroblast‐adipose‐like cell line). Growth and differentiation of 3T3‐L1 cells were monitored by analyzing lipid accumulation and expression of peroxisome proliferator‐activated receptor (PPAR)‐γ mRNA. The medium conditioned by 3T3‐L1 cells was added to J774.1 cells and the cytokines in the supernatant analyzed. Compared with that of cells exposed to a PBS‐conditioned medium, lipid accumulation in 3T3‐L1 cells was significantly suppressed in a dose‐dependent manner by each medium that had been conditioned with LGG and TMC0356. PPAR‐γ mRNA expression in 3T3‐L1 cells was also significantly downregulated (P < 0.01, P < 0.05, respectively). The conditioned medium of 3T3‐L1 adipose phenotype significantly stimulated production of IL‐6 and IL‐12 in J774.1 cells treated with LGG and TMC0356. These results suggest that lactobacilli may suppress differentiation of preadipocytes through macrophage activation and alter the immune responses of macrophages to adipose cells.