The SskA and SrrA response regulators are implicated in oxidative stress responses of hyphae and asexual spores in the phosphorelay signaling network of Aspergillus nidulans

Histidine-to-Aspartate (His-Asp) phosphorelay (or two-component) systems are common signal transduction mechanisms implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. For a model filamentous fungi, Aspergillus nidulans, in this study we first compiled a complete list of His-Asp phosphorelay components, including 15 genes for His-kinase (HK), four genes for response regulator (RR), and only one for histidine-containing phosphotransfer intermediate (HPt). For these RR genes, a set of deletion mutants was constructed so as to create a null allele for each. When examined these mutant strains under various conditions stressful for hyphal growth and asexual spore development, two of them (designated DeltasskA and DeltasrrA) showed a marked phenotype of hypersensitivity to oxidative stresses (particularly, to hydrogen peroxide). In this respect, expression of the vegetative-stage specific catB catalase gene was severely impaired in both mutants. Furthermore, conidia from DeltasskA were hypersensitive not only to treatment with H(2)O(2), but also to treatment at aberrantly low (4 degrees C) and high (50 degrees C) temperatures, resulting in reduced germination efficiency. In this respect, not only the catA catalase gene specific for asexual development, but also a set of genes encoding the enzymes for synthesis of certain stress tolerant compatible solutes, such as trehalose and glycerol, were markedly downregulated in conidia from DeltasskA. These results together are indicative of the physiological importance of the His-Asp phosphorelay signaling network involving the SskA and SrrA response regulators.     

Loss of μ-crystallin causes PPARγ activation and obesity in high-fat diet-fed mice

The thyroid hormone-binding protein μ-crystallin (CRYM) mediates thyroid hormone action by sequestering triiodothyronine in the cytoplasm and regulating the intracellular concentration of thyroid hormone. As thyroid hormone action is closely associated with glycolipid metabolism, it has been proposed that CRYM may contribute to this process by reserving or releasing triiodothyronine in the cytoplasm. We aimed to clarify the relationship between CRYM and glycolipid metabolism by comparing wild-type and CRYM knockout mice fed a high-fat diet. Each group was provided a high-fat diet for 10 weeks, and then their body weight and fasting blood glucose levels were measured. Although no difference in body weight was observed between the two groups with normal diet, the treatment with a high-fat diet was found to induce obesity in the knockout mice. The knockout group displayed increased dietary intake, white adipose tissue, fat cell hypertrophy, and hyperglycemia in the intraperitoneal glucose tolerance test. In CRYM knockout mice, liver fat deposits were more pronounced than in the control group. Enhanced levels of PPARγ, which is known to cause fatty liver, and ACC1, which is a target gene for thyroid hormone and is involved in the fat synthesis, were also detected in the livers of CRYM knockout mice. These observations suggest that CRYM deficiency leads to obesity and lipogenesis, possibly in part through increasing the food intake of mice fed a high-fat diet.     

Diapause hormone directly stimulates the prothoracic glands of diapause larvae under juvenile hormone regulation in the bamboo borer,Omphisa fuscidentalis Hampson

Larval diapause in many lepidopteran insects is induced and maintained by high juvenile hormone (JH). In the case of the bamboo borer, Omphisa fuscidentalis, the effect of JH is the opposite: The application of juvenile hormone analog (JHA: S‐methoprene) terminates larval diapause, unlike in other insect species. Here, we analyzed the expression of JH‐receptor Met, DH‐PBAN, and Kr‐h1 in the subesophageal ganglion (SG) from October to April using semi‐quantitative polymerase chain reaction (PCR). The results show that OfMet and OfDH‐PBAN messenger RNA in the SG are mainly expressed during the larval diapause stage, while OfKr‐h1 increases during the pupal stage. Using tissue culture techniques and an enzyme‐linked immunosorbent assay (ELISA), diapause hormone (DH) was found to induce ecdysteroidogenesis in the culture medium of the prothoracic gland (PG) after incubation for 30 min with 25 ng and 50 ng of DH. Thus, DH is a novel stimulator for the PG. We identified a DHR homolog in the bamboo borer and confirmed that it is expressed in the PG. In addition, for in vitro experiments, DH increased the expression levels of OfDHR, OfEcR‐A, and ecdysone‐inducible genes in the PG. These results demonstrate that DH can function as a prothoracicotropic factor, and this function of DH might be through of DHR expressed on PG cells. Consequently, DH is one of the key factors in larval diapause break which is triggered by JH in the bamboo borer, O. fuscidentalis.     

A food-supply device for maintaining Cotesia vestalis,a larval parasitoid of the diamondback moth Plutella xylostella,in greenhouses

The limited availability of sugar sources (e.g., flowers) in greenhouses may affect biological pest control by parasitoid wasps. However, few studies have focused on feeding devices to provide parasitoids with sugar foods. We investigated the accessibility of a yellow-colored bottle-type feeding device to adult Cotesia vestalis (Halliday), a larval endoparasitoid of the diamondback moth Plutella xylostella (L.). All parasitoids died within four days in a room with no sugar source, whereas 66.7 % of individuals survived if a bottle-type feeding device providing honey solution was installed. We also investigated female longevity in response to different sugar solutions presented in a bottle-type feeding device. Honey and sugar mixtures (glucose and fructose) improved female longevity (38.4 and 39.2 days, respectively) much more than water (3.1 days), indicating these feeding devices containing sugar foods to be potentially useful for maintaining C. vestalis in greenhouses where natural food sources are limited.     

Characterization of Novel MSX1 Mutations Identified in Japanese Patients with Nonsyndromic Tooth Agenesis

Since MSX1 and PAX9 are linked to the pathogenesis of nonsyndromic tooth agenesis, we performed detailed mutational analysis of these two genes sampled from Japanese patients. We identified two novel MSX1 variants with an amino acid substitution within the homeodomain; Thr174Ile (T174I) from a sporadic hypodontia case and Leu205Arg (L205R) from a familial oligodontia case. Both the Thr174 and Leu205 residues in the MSX1 homeodomain are highly conserved among different species. To define possible roles of mutations at these amino acids in the pathogenesis of nonsyndromic tooth agenesis, we performed several functional analyses. It has been demonstrated that MSX1 plays a pivotal role in hard tissue development as a suppressor for mesenchymal cell differentiation. To evaluate the suppression activity of the variants in mesenchymal cells, we used the myoD-promoter, which is one of convenient reporter assay system for MSX1. Although the gene products of these MSX1 variants are stable and capable of normal nuclear localization, they do not suppress myoD-promoter activity in differentiated C2C12 cells. To clarify the molecular mechanisms underlying our results, we performed further analyses including electrophoretic mobility shift assays, and co-immunoprecipitation assays to survey the molecular interactions between the mutant MSX1 proteins and the oligonucleotide DNA with MSX1 consensus binding motif or EZH2 methyltransferase. Since EZH2 is reported to interact with MSX1 and regulate MSX1 mediated gene suppression, we hypothesized that the T174I and L205R substitutions would impair this interaction. We conclude from the results of our experiments that the DNA binding ability of MSX1 is abolished by these two amino acid substitutions. This illustrates a causative role of the T174I and L205R MSX1 homeodomain mutations in tooth agenesis, and suggests that they may influence cell proliferation and differentiation resulting in lesser tooth germ formation in vivo.     

Direct long-term observation of kinesin processivity at low load

The hand-over-hand stepping mechanism of kinesin at low loads is inadequately understood because the number of molecular steps taken per encounter with the microtubule is difficult to measure: optical traps do not register steps at zero load, while evanescent wave microscopy of single molecules of GFP-kinesin suffers from premature photobleaching. Obtaining low-load data is important because it can efficiently distinguish between alternative proposed mechanisms for molecular walking. We report a novel experiment that records the missing data. We fused kinesin to gelsolin, creating a construct that severs and caps rhodamine-phalloidin actin filaments, setting exactly one kinesin molecule on one end of each fluorescent actin filament. Single kinesin molecules labeled in this way can be tracked easily and definitively using a standard epifluorescence microscope. We use the new system to show that, contrary to a recent report, kinesin run length at low load is independent of ATP concentration in the muM to mM range of ATP concentration. Adding competitor ADP in the presence of saturating ATP decreases both velocity and run length. Based on these data, we propose a simplified model for the mechanism of processive stepping.