Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses

Shigella effectors injected into the host cell via the type III secretion system are involved in various aspects of infection. Here, we show that one of the effectors, IpaH9.8, plays a role in modulating inflammatory responses to Shigella infection. In murine lung infection model, DeltaipaH9.8 mutant caused more severe inflammatory responses with increased pro-inflammatory cytokine production levels than did wild-type Shigella, which resulted in a 30-fold decrease in bacterial colonization. Binding assays revealed that IpaH9.8 has a specific affinity to U2AF(35), a mammalian splicing factor, which interferes with U2AF(35)-dependent splicing as assayed for IgM pre-mRNA. Reducing the U2AF(35) level in HeLa cells and infecting HeLa cells with wild-type caused a decrease in the expression of the il-8, RANTES, GM-CSF, and il-1beta genes as examined by RT-PCR. The results indicate that IpaH9.8 plays a role in Shigella infection to optimize the host inflammatory responses, thus facilitating bacterial colonization within the host epithelial cells.     

Proteomic analysis revealed a novel synaptic proline-rich membrane protein (PRR7) associated with PSD-95 and NMDA receptor

Proteomic analyses have revealed a novel synaptic proline-rich membrane protein: PRR7 (proline rich 7), in the postsynaptic density (PSD) fraction of rat forebrain. PRR7 is 269 amino acid residues long, and displays a unique architecture, composed of a very short N-terminal extracellular region, a single membrane spanning domain, and a cytoplasmic domain possessing a proline-rich sequence and a C-terminal type-1 PDZ binding motif. A fraction of PRR7 accumulates in spines along with synapse maturation, and colocalizes with PSD-95 in a punctate pattern in rat hippocampal neural cultures. Immunoprecipitation and GST pull-down assays demonstrated that PRR7 binds to the third PDZ domain of PSD-95. In addition, the NMDA receptor subunits, NR1 and NR2B, specifically co-immunoprecipitated with PRR7. These results suggest that PRR7 is involved in modulating neural activities via interactions with the NMDA receptor and PSD-95, and PSD core formation.     

Nerve-dependent and -independent events in blastema formation during Xenopus froglet limb regeneration

Blastema formation, the initial stage of epimorphic limb regeneration in amphibians, is an essential process to produce regenerates. In our study on nerve dependency of blastema formation, we used forelimb of Xenopus laevis froglets as a system and applied some histological and molecular approaches in order to determine early events during blastema formation. We also investigated the lateral wound healing in comparison to blastema formation in limb regeneration. Our study confirmed at the molecular level that there are nerve-dependent and -independent events during blastema formation after limb amputation, Tbx5 and Prx1, reliable markers of initiation of limb regeneration, that start to be expressed independently of nerve supply, although their expressions cannot be maintained without nerve supply. We also found that cell proliferation activity, cell survival and expression of Fgf8, Fgf10 and Msx1 in the blastema were affected by denervation, suggesting that these events specific for blastema outgrowth are controlled by the nerve supply. Wound healing, which is thought to be categorized into tissue regeneration, shares some nerve-independent events with epimorphic limb regeneration, although the healing process results in simple restoration of wounded tissue. Overall, our results demonstrate that dedifferentiated blastemal cells formed at the initial phase of limb regeneration must enter the nerve-dependent epimorphic phase for further processes, including blastema outgrowth, and that failure of entry results in a simple redifferentiation as tissue regeneration.     

Social stress decreases marking behavior independently of testosterone in Mongolian gerbils

This study examined the endogenous androgen regulation of the marking behavior in Mongolian gerbils (Meriones unguiculatus). In the first experiment, developmental changes of fecal testosterone levels, ventral gland growth, and the marking frequency of male gerbils were investigated. From 9 weeks of age, marking frequency increased with increases in fecal testosterone levels and ventral gland size. The ventral gland size and marking frequency were significantly correlated to the fecal testosterone level. In the second experiment, we hypothesized that reduction in the marking frequency of subordinate males after social confrontations was controlled by a decrease in the circulating testosterone level, and we followed changes in marking frequency, endocrine status, and ventral gland size after social confrontations in which two adult male gerbils established their social ranks by fighting. As expected, marking frequency and ventral gland size were significantly related to social rank, that is, marking frequency was higher among dominant gerbils and lower among subordinates. In addition, fecal corticosterone levels among subordinates were higher than those of dominant animals. However, neither the fecal and plasma testosterone levels, nor testis size, differed between dominant and subordinate gerbils. These results revealed that endogenous androgen played a role in regulating marking behavior and ventral gland size during the developmental stage and that the reductions in marking frequency and ventral gland size occurring in subordinate males after social confrontations were not directly regulated by androgen changes.     

An Arginine Specific Protease from Spirulina platensis

An arginine specific protease, Sp-protease, was purified by column chromatography from freeze-dried Spirulina platensis using a five-step process. Purified Sp-protease has a molecular weight of 80 kDa. It hydrolyzed the synthetic substrates containing arginine residue in the P1 position but did not hydrolyze synthetic substrates containing other amino acid residues, including lysine residue in the P1 position. Among the synthetic substrates tested, a substrate of plasminogen activator (Pyr-Gly-Arg-MCA) was hydrolyzed most effectively with the enzyme (K_m = 5.5 × 10⁻⁶ M), and fibrin gel was solubilized via activation of intrinsic plasminogen to plasmin with the enzyme. Activity was inhibited completely with camostat mesilate (K_i = 1.1 × 10⁻⁸ M) and leupeptin (K_i = 3.9 × 10⁻⁸ M) but was not inhibited with N^α-tosyl-L-lysine chloromethyl ketone (TLCK). The optimum pH of the enzyme has a range of pH 9.0 to pH 11.0. The optimum temperature was 50°C; the enzyme was stable at 0–50°C.     

A population genetic model for lateral dimorphism frequency in fishes

Two morphological types, righty and lefty, are found in several fish species. Righty predators mainly prey on lefty prey and vice versa (called cross predation). This dimorphism is heritable in a Mendelian one-locus–two-allele fashion. The frequency of righty individuals in a population oscillates periodically. To determine the effect of cross predation on this oscillation, we constructed mathematical models that describe a genetic basis of lateral dimorphism assuming genetic drift in a one-predator–two-prey system and three trophic levels with omnivory. Both models have an equilibrium of both righty and lefty types at a frequency of 0.5. Mathematical analyses and computer simulations showed that the dimorphism is maintained and that its frequency oscillated with or without genetic drift. Large degrees of drift and high intrinsic growth rates and predation efficiencies of prey species caused the frequency oscillations to be amplified and their period to be long. When cross predation decreased as a proportion of all predation, the righty frequency of a prey species fluctuated non-periodically. These differences in fluctuation patterns were observed in natural systems. Our model suggests that both cross predation and genetic drift dictate the maintenance of dimorphism and the patterns of its fluctuations.     

Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production in the transgenic Arabidopsis thaliana by the in vitro evolved highly active mutants of polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae

In this study, the enhancement of photosynthetic PHA production was achieved using the highly active mutants of PHA synthase created by the in vitro evolutionally techniques. The wild-type and mutated PHA synthase genes from Aeromonas caviae were introduced into Arabidopsis thaliana together with the NADPH-dependent acetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, led to an 8-10-fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content was further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymers consisting of 0.2-0.8 mol % 3HV. The monomer composition of the P(3HB-co-3HV) copolymers synthesized by the wild-type and mutated PHA synthases reflected the substrate specificities observed in Escherichia coli. These results indicate that in vitro evolved PHA synthases can enhance the productivity of PHA and regulate the monomer composition in transgenic plants.     

Disintegration of layer-by-layer assemblies composed of 2-iminobiotin-labeled poly(ethyleneimine) and avidin

A layer-by-layer thin film composed of avidin and 2-iminobiotin-labeled poly(ethyleneimine) (ib-PEI) was prepared and their sensitivity to the environmental pH and biotin was studied. The avidin/ib-PEI multilayer assemblies were stable at pH 8-12, whereas the assemblies were decomposed at pH 5-6 due to the low affinity of the protonated iminobiotin residue to avidin. The avidin/ib-PEI assemblies can be disintegrated upon addition of biotin and analogues in the solution as a result of the preferential binding of biotin or analogues to the binding site of avidin. The decomposition rate was arbitrarily controlled by changing the type of stimulant (biotin or analogues) and its concentration. The avidin/ib-PEI assemblies were disintegrated rapidly by the addition of biotin or desthiobiotin, whereas the rate of decomposition was rather slow upon addition of lipoic acid or 2-(4'-hydroxyphenylazo)benzoic acid. The present system may be useful for constructing the stimuli-sensitive devices that can release drug or other functional molecules.     

Class I and class II chitin synthases are involved in septum formation in the filamentous fungus Aspergillus nidulans

The class II and class I chitin synthases of the filamentous fungus Aspergillus nidulans are encoded by chsA and chsC, respectively. Previously, we presented several lines of evidence suggesting that ChsA and ChsC have overlapping functions in maintaining cell wall integrity. In order to determine the functions of these chitin synthases, we employed electron and fluorescence microscopy and investigated in detail the cell wall of a DeltachsA DeltachsC double mutant (DeltaAC mutant) along with the localization of ChsA and ChsC. In the lateral cell wall of the DeltaAC mutant, electron-transparent regions were thickened. Septa of the DeltaAC mutant were aberrantly thick and had a large pore. Some septa were located abnormally close to adjacent septa. A functional hemagglutinin (HA)-tagged ChsA (HA-ChsA) and a functional FLAG-tagged ChsC (FLAG-ChsC) were each localized to a subset of septation sites. Comparison with the localization pattern of actin, which is known to localize at forming septa, suggested that ChsA and ChsC transiently exist at the septation sites during and shortly after septum formation. Double staining of HA-ChsA and FLAG-ChsC indicated that their localizations were not identical but partly overlapped at the septation sites. Fluorescence of FLAG-ChsC, but not of HA-ChsA, was also observed at hyphal tips. These data indicate that ChsA and ChsC share overlapping roles in septum formation.     

Molecular mechanisms associated with leptin resistance: n-3 polyunsaturated fatty acids induce alterations in the tight junction of the brain

High-fat diets cause peripheral leptin resistance, and dietary lipid composition affects sensitivity to leptin. We examined the role of n-3 polyunsaturated fatty acid (PUFA) in peripheral leptin resistance. Dietary PUFAs (0.4% wt/wt) caused insensitivity to peripherally but not intracerebroventricularly administered leptin. n-3 PUFA increased body weight, associated with a significant reduction of leptin concentration in the cerebrospinal fluid. Dietary n-3 PUFA reduced transport of endogenous or exogenously administered leptin into the brain, associated with increased expression of hypothalamic occludin, but caused no change in expression of leptin receptors, proteins associated with leptin signaling or other tight junction proteins. Continuous intracerebroventricular infusion of an antisense morpholino oligonucleotide targeted to occludin mRNA reversed n-3 PUFA-induced insensitivity to peripherally administered leptin. We conclude that n-3 PUFA induces peripheral leptin resistance via an increase in the expression of hypothalamic occludin, reducing paracellular transport of leptin into the brain.