4-Acylamino-6-arylfuro[2,3-d]pyrimidines: potent and selective glycogen synthase kinase-3 inhibitors

Modeling studies of a furo[2,3-d]pyrimidine GSK-3 hit compound 1 superimposed onto the X-ray crystal structure of a legacy pyrazolo[3,4-c]pyridazine GSK-3 inhibitor 2 led to the identification of 4-acylamino-6-arylfuro[2,3-d]pyrimidine template 3. Synthesis of analogues based on template 3 has resulted in a number of potent and selective GSK-3beta inhibitors. The most potent and selective compound was the m-pyridyl analogue 24.     

Structure of the trypsin-binding domain of Bowman-Birk type protease inhibitor and its interaction with trypsin

The crystal structure of the complex formed by bovine trypsin and Bowman-Birk type protease inhibitor AB-I extracted from azuki beans (Vigna angularis) 'Takara' has been analyzed. The structure was solved by the application of the phase combination of single isomorphous phases and trypsin model phases, followed by phase improvement using the iterative Fourier technique. From the resulting electron density map, a three-dimensional atomic model of the trypsin binding domain of AB-I has been built. The peptide chain at the trypsin reactive site turns back sharply at Pro29 and forms a 9-residue ring (Cys24-Cys32). The 'front side' of this ring, consisting of the reactive site (Cys24-Met28), interacts with trypsin in a similar manner to other families of inhibitors and forms a stable complex, which seems to be maintained by the interactions with the 'back side' of this ring (Pro29-Cys34). The similar spatial arrangements of the 'back side' of this inhibitor and the 'secondary contact region' of the other inhibitors with respect to the reactive site suggest an important common role of these regions in exhibiting inhibitory activity.     

Distribution and dietary relationships of the Japanese temperate bass Lateolabrax japonicus juveniles with two contrasting copepod assemblages in estuarine nursery grounds in the Ariake Sea, Japan

Early juvenile Japanese temperate bass Lateolabrax japonicus samples were collected during four cruises to study the spatial and temporal patterns of distribution and feeding habits of the fish in Chikugo estuary, Ariake Sea, Japan. Gut contents were studied by separating, identifying and counting the prey organisms. Plankton samples were collected during each cruise to study the numerical abundance of copepods in the water. Copepod dry biomass and gut content dry mass were estimated. Juveniles were distributed over wide estuarine areas in salinities ranging from as low as 0·37 to as high as 28·81. Considerable spatial and temporal variations were observed in the copepod distribution in the environment and in the food habits of the fish. Two distinctly different copepod assemblages were identified along the estuary: one in the upper river, dominated by a single species Sinocalanus sinensis, and the other a multi‐specific assemblage in the lower estuary dominated by common coastal copepods such as Acartia omorii, Oithona davisae, Paracalanus parvus and Calanus sinicus. The gut content composition corresponded strongly with the copepod composition in the environment, i.e. a single species (S. sinensis) dietary habit at the upper river and a multi‐specific dietary habit dominated by the common coastal copepods in the lower estuary. Ivlev's electivity index showed that the fish strongly preferred larger copepods and avoided smaller ones. Higher dry biomass of copepods in the water as well as higher dry masses of the gut contents were recorded in the low‐to‐medium saline upper river areas, indicating that these areas are of particular importance as nursery grounds for the juvenile Japanese temperate bass. It was speculated that ascending to the upstream nursery areas to utilize S. sinensis, which is the single dominant copepod in these areas, is one of the most important survival strategies of the Japanese temperate bass juveniles in the Chikugo estuary.     

Isolation of B subunit‐specific monoclonal antibody clones that strongly neutralize the toxicity of Shiga toxin 2

Shiga toxin 2 (Stx2)‐specific mAb‐producing hybridoma clones were generated from mice. Because mice tend to produce small amounts of B subunit (Stx2B)‐specific antibodies at the polyclonal antibody level after immunization via the parenteral route, mice were immunized intranasally with Stx2 toxoids with a mutant heat‐labile enterotoxin as a mucosal adjuvant; 11 different hybridoma clones were obtained in two trials. Six of them were A subunit (Stx2A)‐specific whereas five were Stx2B‐specific antibody‐producing clones. The in vitro neutralization activity of Stx2B‐specific mAbs against Stx2 was greater than that of Stx2A‐specific mAbs on HeLa229 cells. Furthermore, even at low concentrations two of the Stx2B‐specific mAbs (45 and 75D9) completely inhibited receptor binding and showed in vivo neutralization activity against a fivefold median lethal dose of Stx2 in mice. In western blot analysis, these Stx2B‐specific neutralization antibodies did not react to three different mutant forms of Stx2, each amino acid residue of which was associated with receptor binding. Additionally, the nucleotide sequences of the V_H and V_L regions of clones 45 and 75D9 were determined. Our Stx2B‐specific mAbs may be new candidates for the development of mouse‐human chimeric Stx2‐neutralizing antibodies which have fewer adverse effects than animal antibodies for enterohemorrhagic Escherichia coli infection.     

Three-dimensional electron microscopy analysis of ndc10-1 mutant reveals an aberrant organization of the mitotic spindle and spindle pole body defects in Saccharomyces cerevisiae

Kinetochore components play a major role in regulating the transmission of genetic information during cell division. Ndc10p, a kinetochore component of the essential CBF3 complex in budding yeast is required for chromosome attachment to the mitotic spindle. ndc10-1 mutant was shown to display chromosome mis-segregation as well as an aberrant mitotic spindle (Goh and Kilmartin, 1993). In addition, Ndc10p localizes along the spindle microtubules (Muller-Reichert et al., 2003). To further understand the role of Ndc10p in the mitotic apparatus, we performed a three-dimensional electron microscopy (EM) reconstruction of mitotic spindles from serial sections of cryo-immobilized ndc10-1 mutant cells. This analysis reveals a dramatic reduction in the number of microtubules present in the half-spindle, which is connected to the newly formed spindle pole body (SPB) in ndc10-1 cells. Moreover, in contrast to wild-type (WT) cells, ndc10-1 cells showed a significantly lower signal intensity of the SPB components Spc42p and Spc110p fused with GFP, in mother cell bodies compared with buds. A subsequent EM analysis also showed clear defects in the newly formed SPB, which remains in the mother cell during anaphase. These results suggest that Ndc10p is required for maturation of the newly formed SPB. Intriguingly, mutations in other kinetochore components, ndc80-1 and spc24-1, showed kinetochore detachment from the spindle, similar to ndc10-1, but did not display defects in SPBs. This suggests that unattached kinetochores are not sufficient to cause SPB defects in ndc10-1 cells. We propose that Ndc10p, alongside its role in kinetochore–microtubule interaction, is also essential for SPB maturation and mitotic spindle integrity.     

Interferon signaling suppresses the unfolded protein response and induces cell death in hepatocytes accumulating hepatitis B surface antigen

Virus infection, such as hepatitis B virus (HBV), occasionally causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR are poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFNα causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFNα mediated liver injury. Analyses of total gene expression and UPR biomarkers’ protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFNα administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFNα mediated liver injury. These results suggest that IFNα induces ER stress-associated cell death by reducing UPR. IFNγ uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism of IFN-mediated cell death. This study also identifies UPR as a potential target for regulating ER stress-associated cell death.     

The role of p63 in embryonic external genitalia outgrowth in mice

Embryonic external genitalia (genital tubercle [GT]) protrude from the cloaca and outgrow as cloacal development progresses. Individual gene functions and knockout phenotypes in GT development have been extensively analyzed; however, the interactions between these genes are not fully understood. In this study, we investigated the role of p63, focusing on its interaction with the Shh–Wnt/Ctnnb1–Fgf8 pathway, a signaling network that is known to play a role in GT outgrowth. p63 was expressed in the epithelial tissues of the GT at E11.5, and the distal tip of the GT predominantly expressed the ΔNp63α isoform. The GTs in p63 knockout embryos had normal Shh expression, but CTNNB1 protein and Fgf8 gene expression in the distal urethral epithelium was decreased or lost. Constitutive expression of CTNNB1 in p63-null embryos restored Fgf8 expression, accompanied by small bud structure development; however, such bud structures could not be maintained by E13.5, at which point mutant GTs exhibited severe abnormalities showing a split shape with a hemorrhagic cloaca. Therefore, p63 is a key component of the signaling pathway that triggers Fgf8 expression in the distal urethral epithelium and contributes to GT outgrowth by ensuring the structural integrity of the cloacal epithelia. Altogether, we propose that p63 plays an essential role in the signaling network for the development of external genitalia.     

Daphnia curvirostris Eylmann in Japanese high mountain waters (Cladocera: Daphniidae)

A species of Daphnia, Daphnia curvirostris Eylmann, found in high mountain lakes and ponds in central Japan is described. Although there were some differences in the shape of the male rostrum and the chromosome number between European populations as described by Johnson (1952) and Trentini (1980), and Japanese ones collected from high mountain waters, Japanese specimens had many characteristics similar to the taxon D. curvirostris of Europe.     

Serotonin differentially regulates short- and long-term prediction of rewards in the ventral and dorsal striatum

Background

The ability to select an action by considering both delays and amount of reward outcome is critical for maximizing long-term benefits. Although previous animal experiments on impulsivity have suggested a role of serotonin in behaviors requiring prediction of delayed rewards, the underlying neural mechanism is unclear.

Methodology/Principal Findings

To elucidate the role of serotonin in the evaluation of delayed rewards, we performed a functional brain imaging experiment in which subjects chose small-immediate or large-delayed liquid rewards under dietary regulation of tryptophan, a precursor of serotonin. A model-based analysis revealed that the activity of the ventral part of the striatum was correlated with reward prediction at shorter time scales, and this correlated activity was stronger at low serotonin levels. By contrast, the activity of the dorsal part of the striatum was correlated with reward prediction at longer time scales, and this correlated activity was stronger at high serotonin levels.

Conclusions/Significance

Our results suggest that serotonin controls the time scale of reward prediction by differentially regulating activities within the striatum.     

Disruption of the murine intestinal alkaline phosphatase gene Akp3 impairs lipid transcytosis and induces visceral fat accumulation and hepatic steatosis

Intestinal alkaline phosphatase (IAP) is involved in the process of fat absorption, a conclusion confirmed by an altered lipid transport and a faster body weight gain from 10 to 30 wk in both male and female mice with a homozygous null mutation of the IAP coding gene (Akp3(-/-) mice). This study was aimed to delineate morphologically and quantitatively the accelerated lipid absorption in male Akp3(-/-) mice. Feeding a corn oil bolus produced an earlier peak of triacylglycerol in serum (2 vs. 4 h for Akp3(-/-) and wild-type mice, respectively) and an approximately twofold increase in serum triacylglycerol concentration in Akp3(-/-) mice injected with a lipolysis inhibitor, Triton WR-1339. A corn oil load induced the threefold enlargement of the Golgi vacuoles in male wild-type mice but not in Akp3(-/-) mice, indicating that absorbed lipids rarely reached the Golgi complex and that the transcytosis of lipid droplets does not follow the normal pathway in male Akp3(-/-) mice. Force feeding an exaggerated fat intake by a 30% fat chow for 10 wk induced obesity in both male Akp3(-/-) and wild-type mice, and therefore no phenotypic difference was observed between the two. On the other hand, the forced high-fat chow induced an 18% greater body weight gain, hepatic steatosis, and visceral fat accumulation in female Akp3(-/-) mice but not in female wild-type controls. These results provide further evidence that IAP is involved in the regulation of the lipid absorption process and that its absence leads to progressive metabolic abnormalities in certain fat-forced conditions.