GFP transgenic mice reveal active canonical Wnt signal in neonatal brain and in adult liver and spleen

In the past decades, the function of the Wnt canonical pathway during embryogenesis has been intensively investigated; however, little survey of neonatal and adult tissues has been made, and the role of this pathway remains largely unknown. To investigate its role in mature tissues, we generated two new reporter transgenic mouse lines, ins-TOPEGFP and ins-TOPGAL, that drive EGFP and beta-galactosidase expression under TCF/beta-catenin, respectively. To obtain the accurate expression pattern, we flanked these transgenes with the HS4 insulator to reduce chromosomal positional effects. Analysis of embryos showed that the reporter genes were activated in regions where canonical Wnt activity has been implicated. Furthermore, their expression patterns were consistent in both lines, indicating the accuracy of the reporter signal. In the neonatal brain, the reporter signal was detected in the mesencephalon and hippocampus. In the adult mice, the reporter signal was found in the mature pericenteral hepatocytes in the normal liver. Furthermore, during inflammation the number of T cells expressing the reporter gene increased in the adult spleen. Thus, in this research, we identified two organs, i.e., the liver and spleen, as novel organs in which the Wnt canonical signal is in motion in the adult. These transgenic lines will provide us broader opportunities to investigate the function of the Wnt canonical pathway in vivo.     

Ontogenic phototactic behaviors of larval stages in intertidal barnacles

Hydrobiologia - During larval development of the intertidal barnacle Fistulobalanus albicostatus, larvae in the naupliar stages I and II (NI&II) possess a single naupliar eye, and later...     

Class I Myosins Have Overlapping and Specialized Functions in Left-Right Asymmetric Development in Drosophila

The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left–right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. Here we generated class I myosin double and triple mutants to address this issue. We found that the triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E, had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Our studies also revealed differential colocalization of the class I myosins with filamentous (F)-actin in the brush border of intestinal enterocytes.     

A highly conserved pocket on PP2A‐B56 is required for hSgo1 binding and cohesion protection during mitosis

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A‐B56 phosphatase through a coiled‐coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A‐B56‐hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A‐B56 substrates containing a canonical B56 binding motif. We find that PP2A‐B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis.     

Development and Application of a Whole-Genome Simple Sequence Repeat Panel for High-Throughput Genotyping in Soybean

Among commonly applied molecular markers, simple sequence repeats (SSRs, or microsatellites) possess advantages such as a high level of polymorphism and codominant pattern of inheritance at individual loci. To facilitate systematic and rapid genetic mapping in soybean, we designed a genotyping panel comprised 304 SSR markers selected for allelic diversity and chromosomal location so as to provide wide coverage. Most primer pairs for the markers in the panel were redesigned to yield amplicons of 80–600 bp in multiplex polymerase chain reaction (PCR) and fluorescence-based sequencer analysis, and they were labelled with one of four different fluorescent dyes. Multiplex PCR with sets of six to eight primer pairs per reaction generated allelic data for 283 of the 304 SSR loci in three different mapping populations, with the loci mapping to the same positions as previously determined. Four SSRs on each chromosome were analysed for allelic diversity in 87 diverse soybean germplasms with four-plex PCR. These 80 loci showed an average allele number and polymorphic information content value of 14.8 and 0.78, respectively. The high level of polymorphism, ease of analysis, and high accuracy of the SSR genotyping panel should render it widely applicable to soybean genetics and breeding.     

Detection of Fas ligand in the bovine oviduct

Presence of a Fas-Fas ligand (FasL) system defines the immune-privileged status of certain tissues such as placenta. This study examined the fluids and tissue(s) of the bovine oviduct, where both spermatozoa and early embryos escape elimination by the female immune system, for the presence and the distribution of Fas and FasL, which might provide an explanation for the immune-privileged site of this organ. In the present study, the immunolocalisation of FasL and Fas, as well as the gene expression of FasL, were determined in the uterotubal junction (UTJ), isthmic (I) and ampullar (A) segments of the oviduct during oestrus and the luteal phase of the oestrous cycle. The degree of apoptosis of oviductal epithelium was examined by the TUNEL method. Oviductal fluid (ODF), collected chronically via indwelling catheters from the I or A segments during both non-luteal and luteal phases of the cycle, was analysed for the presence of FasL. The Fas immunostaining was scattered along the epithelium of all regions of the oviduct and cycle stages investigated, whereas FasL immunolabelling was more conspicuous in oestrous samples. This staining disappeared during the luteal phase, which was particularly evident in the sperm reservoir (UTJ and I). There were fewer TUNEL-positive cells than Fas- or FasL-positive cells in the oviductal epithelium, suggesting that tubal Fas and FasL are not directly involved in epithelial apoptosis. Western blot analyses detected FasL in ODF collected from both I and A, most conspicuously as a 24-27kDa band but also at a 40-45kDa band level. FasL mRNA was expressed in the epithelial cells from the sperm reservoir and A during both non-luteal and luteal phases. However, the level of expression differed significantly between segments during the luteal phase. The results provide novel evidence that the Fas-FasL system is present in the bovine oviduct and could be involved in mediating survival of spermatozoa and early embryos.     

Quantitative analysis of cereulide, an emetic toxin of Bacillus cereus, by using rat liver mitochondria

An emetic toxin cereulide, produced by Bacillus cereus, causes emetic food poisonings, but a method for quantitative measurement of cereulide has not been well established. A current detection method is a bioassay method using the HEp-2 cell vacuolation test, but it was unable to measure an accurate concentration. We established a quantitative assay for cereulide based on its mitochondrial respiratory uncoupling activity. The oxygen consumption in a reaction medium containing rat liver mitochondria was rapid in the presence of cereulide. Thus uncoupling effect of cereulide on mitochondrial respiration was similar to those of uncouplers 2,4-dinitrophenol (DNP), carbonylcyanide m-chlorophenylhydrazone (CCCP), and valinomycin. This method gave constant results over a wide range of cereulide concentrations, ranging from 0.05 to 100 microg/ml. The minimum cereulide concentration to detect uncoupled oxygen consumption was 50 ng/ml and increased dose-dependently to the maximum level. Semi-log relationship between the oxygen consumption rate and the cereulide concentration enables this method to quantify cereulide. The results of this method were highly reproducible as compared with the HEp-2 cell vacuolation test and were in good agreement with those of the HEp-2 cell vacuolation test. The enterotoxin of B. cereus or Staphylococcus aureus did not show any effect on the oxygen consumption, indicating this method is specific for the identification of cereulide as a causative agent of emetic food poisonings.