Phospholipase Cε, an Effector of Ras and Rap Small GTPases,Is Required for Airway Inflammatory Response in a Mouse Model of Bronchial Asthma

Background

Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases and expressed in non-immune cells. It is well established that PLCε plays an important role in skin inflammation, such as that elicited by phorbol ester painting or ultraviolet irradiation and contact dermatitis that is mediated by T helper (Th) 1 cells, through upregulating inflammatory cytokine production by keratinocytes and dermal fibroblasts. However, little is known about whether PLCε is involved in regulation of inflammation in the respiratory system, such as Th2-cells-mediated allergic asthma.

Methods

We prepared a mouse model of allergic asthma using PLCε ^+/+ mice and PLCε ^ΔX/ΔX mutant mice in which PLCε was catalytically-inactive. Mice with different PLCε genotypes were immunized with ovalbumin (OVA) followed by the challenge with an OVA-containing aerosol to induce asthmatic response, which was assessed by analyzing airway hyper-responsiveness, bronchoalveolar lavage fluids, inflammatory cytokine levels, and OVA-specific immunoglobulin (Ig) levels. Effects of PLCε genotype on cytokine production were also examined with primary-cultured bronchial epithelial cells.

Results

After OVA challenge, the OVA-immunized PLCε ^ΔX/ΔX mice exhibited substantially attenuated airway hyper-responsiveness and broncial inflammation, which were accompanied by reduced Th2 cytokine content in the bronchoalveolar lavage fluids. In contrast, the serum levels of OVA-specific IgGs and IgE were not affected by the PLCε genotype, suggesting that sensitization was PLCε-independent. In the challenged mice, PLCε deficiency reduced proinflammatory cytokine production in the bronchial epithelial cells. Primary-cultured bronchial epithelial cells prepared from PLCε ^ΔX/ΔX mice showed attenuated pro-inflammatory cytokine production when stimulated with tumor necrosis factor-α, suggesting that reduced cytokine production in PLCε ^ΔX/ΔX mice was due to cell-autonomous effect of PLCε deficiency.

Conclusions

PLCε plays an important role in the pathogenesis of bronchial asthma through upregulating inflammatory cytokine production by the bronchial epithelial cells.     

Structure and physicochemical properties of starches from kidney bean seeds at immature,premature and mature stages of development

Starches from kidney bean (Phaseolus vulgaris L. cv. Toramame) seeds at the immature, premature, mature stages of development were examined. The starch content increased from 94, 219 to 265 mg per seed. Starches showed the C(a)-crystalline type composed of small (<5 micrometer) and large (10-35 micrometer) granules, with the large granules largely increasing with maturity. The amylose content increased from 21, 26 to 27%, and rapid viscograms and DSC thermograms suggested that the mature-stage starch was gelatinized with ease. The amylose increased in size from DPn 820, 1000 to 1080 and a number of chains per molecule (NC) from 3.3, 4.2 to 4.5. The branched amylose was a minor component (11-18% by mole) with NC 20-22. The amylopectin was similar in CL (23), beta-amylolysis limit (59%), and chain-length distribution, but reduced in size (DPn 17,100-5270) and increased in content of phosphorus (114-174 ppm) with an increase in the amount of phosphorus linked to C-6 of the glucose residue (8-66%).     

Differentiation in wild-type allele of the sd1 locus concerning culm length between indica and japonica subspecies of Oryza sativa (rice)

A dwarfing gene (allele) sd1-d has been intensively utilized to develop short-culm indica varieties in southeast Asia up to now. Before the first sd1-d-carrying variety IR8 was released, rice researchers had recognized the general tendency that culm length is higher in indica varieties than in temperate-japonica ones. Inter-subspecific difference of the tall (wild-type) allele SD1 at the sd1 locus was examined on the common genetic background, using five isogenic lines developed by substituting sd1-d of the recurrent parent IR36 by SD1s of two indica varieties, two temperate-japonica varieties and one tropical-japonica variety. The two indica -donor isogenic lines had longer culms than the three japonica-donor isogenic lines consistently in two different environmental conditions. Moreover, nonsynonymous single-nucleotide polymorphism between the two subspecies was detected at two sites in Exon 1 and Exon 3 of the sd1 locus. It is demonstrated that the inter-subspecific differentiation of SD1 contributes height difference between indica and japonica. The indica-originating and japonica-originating alleles at the sd1 locus were designated as SD1-in(t) and SD1-ja(t), respectively.     

l-NAME inhibits tumor cell progression and pulmonary metastasis of r/m HM-SFME-1 cells by decreasing NO from tumor cells and TNF-α from macrophages

Highly metastatic ras/myc-transformed serum-free mouse embryo (r/m HM-SFME-1) cells were injected subcutaneously to mice and the effects of Nω-nitro-l-arginine methyl ester (l-NAME) on the tumor progression and pulmonary metastasis were investigated. In addition, production of nitric oxide (NO), matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-α) in the tumor cells and in a mouse macrophage-like cell line, J774.1 cells, was analyzed. The increase in footpad thickness was significantly smaller in the mice which were fed the l-NAME containing water (4.24 ± 0.39 mg/day/mouse). The number of the tumor cells metastasized to the lungs was smaller in the l-NAME treated mice, although statistical significance was not found. Co-treatment of r/m HM-SFME-1 cells with interferon-gamma (IFN-γ; 100 U/ml) and lipopolysaccharide (LPS; 0.5 μg/ml) significantly enhanced NO production, and the presence of l-NAME at 1 mM significantly decreased this response. In r/m HM-SFME-1 cells, MMP-2 was undetectable and MMP-9 was also very little in the basal level, and both MMPs were unaffected by the IFN-γ and/or LPS treatments, not to mention by the l-NAME treatment. In J774.1 cells, any treatment including LPS appeared to enhance MMP-9 production, however, this upregulation was not inhibited by the additional presence of l-NAME. Production of TNF-α by J774.1 cells was markedly enhanced with LPS treatment, and this enhancement was significantly reduced in the presence of l-NAME. These results indicate that the inhibitory effects of l-NAME on the tumor cell progression and pulmonary metastasis could be due to suppression of NO from tumor cells and TNF-α from macrophages (Mol Cell Biochem, 2007). Hideaki Yamaguchi and Yumi Kidachi contributed equally to this work.     

Morphological analyses and 3D modeling of the tongue musculature of the chimpanzee (Pan troglodytes)

Knowledge of the comparative anatomy of tongue musculature is crucial to the discussion of the origin and the evolution of speech because of the indispensable role played by this organ in speech. However, the tongue musculature of primates has rarely been studied. In a previous study, the author analyzed human tongue musculature and developed a 3D model of this organ [Takemoto, Journal of Speech, Language, and Hearing Research 44:95-107, 2001]. In this study, the tongue musculature of chimpanzees was examined using methods similar to those used for humans. Results showed that tongue musculature was topologically the same for both humans and chimpanzees. As in humans, the tongue musculature of chimpanzees consisted of inner and outer regions. The inner musculature was composed of serial "structural units," made up of two types of laminae whose fibers were perpendicular to the tongue surface. The outer musculature was a thin layer of fibers oriented parallel to the surface and superficial to the inner musculature. Although the tongue musculature of humans and chimpanzees is similar, the external shapes differ: the chimpanzee tongue is flat, whereas the human tongue is round. Applying the muscular hydrostat theory to the external shape of the tongue suggests that the primary actions of the chimpanzee tongue are protrusion and retrusion, whereas the human tongue can be deformed in the oral cavity with a high degree of freedom. It is hypothesized that the evolution of the external shape of the tongue is one of the factors that led to the development of human speech. The results of this study suggest that modeling based on muscular hydrostatic theory of the effects of changes in external tongue shape on articulatory movements should be included in discussions on the origin of speech.     

Expression of gamma-glutamyltransferase 1 in glioblastoma cells confers resistance to cystine deprivation–induced ferroptosis

Ferroptosis is an iron-dependent mode of cell death caused by excessive oxidative damage to lipids. Lipid peroxidation is normally suppressed by glutathione peroxidase 4, which requires reduced glutathione. Cystine is a major resource for glutathione synthesis, especially in cancer cells. Therefore, cystine deprivation or inhibition of cystine uptake promotes ferroptosis in cancer cells. However, the roles of other molecules involved in cysteine deprivation–induced ferroptosis are unexplored. We report here that the expression of gamma-glutamyltransferase 1 (GGT1), an enzyme that cleaves extracellular glutathione, determines the sensitivity of glioblastoma cells to cystine deprivation–induced ferroptosis at high cell density (HD). In glioblastoma cells expressing GGT1, pharmacological inhibition or deletion of GGT1 suppressed the cell density–induced increase in intracellular glutathione levels and cell viability under cystine deprivation, which were restored by the addition of cysteinylglycine, the GGT product of glutathione cleavage. On the other hand, cystine deprivation induced glutathione depletion and ferroptosis in GGT1-deficient glioblastoma cells even at an HD. Exogenous expression of GGT1 in GGT1-deficient glioblastoma cells inhibited cystine deprivation–induced glutathione depletion and ferroptosis at an HD. This suggests that GGT1 plays an important role in glioblastoma cell survival under cystine-limited and HD conditions. We conclude that combining GGT inhibitors with ferroptosis inducers may provide an effective therapeutic approach for treating glioblastoma.     

Gene flow of Acanthaster planci (L.) in relation to ocean currents revealed by microsatellite analysis

Population outbreaks of the coral-eating starfish, Acanthaster planci , are hypothesized to spread to many localities in the Indo-Pacific Ocean through dispersal of planktonic larvae. To elucidate the gene flow of A. planci across the Indo-Pacific in relation to ocean currents and to test the larval dispersal hypothesis, the genetic structure among 23 samples over the Indo-Pacific was analysed using seven highly polymorphic microsatellite loci. The F -statistics and genetic admixture analysis detected genetically distinct groups in accordance with ocean current systems, that is, the Southeast African group (Kenya and Mayotte), the Northwestern Pacific group (the Philippines and Japan), Palau, the North Central Pacific group (Majuro and Pohnpei), the Great Barrier Reef, Fiji, and French Polynesia, with a large genetic break between the Indian and Pacific Oceans. A pattern of significant isolation by distance was observed among all samples ( P =  0.001, r  = 0.88, n  = 253, Mantel test), indicating restricted gene flow among the samples in accordance with geographical distances. The data also indicated strong gene flow within the Southeast African, Northwestern Pacific, and Great Barrier Reef groups. These results suggest that the western boundary currents have strong influence on gene flow of this species and may trigger secondary outbreaks.     

Invasions by ladybugs,ladybirds, and other predatory beetles

Species of predatory Coleoptera have become abundant in new geographic regions recently, raising concerns for invaded ecosystems. We address this topic by focusing on invasive alien ladybird beetles (Coccinellidae; known also as ladybugs). Humans appear directly or indirectly responsible for all or most ladybird invasions. Factors hypothesized to have promoted ladybird invasions include genetic diversity (e.g., for polymorphism), phenotypic plasticity, adaptation and genetic shift, generalized diet and habitat preferences, flexible life history and reproduction, large body size, and release from enemies. Factors such as climate, habitat and prey availability, and biotic resistance may sometimes prevent or slow ladybird invasions. Indigenous species (e.g., herbivores) may suffer from invasions, and biological control programs may be affected. Species of indigenous ladybirds throughout the world are reported to have declined in abundance following ladybird invasions, with increased competition and/or intraguild predation most often hypothesized or inferred. Similar recent studies especially of ground beetles (Carabidae) also make clear the potential of invasive alien predatory Coleoptera to disrupt invaded natural and agricultural ecosystems.     

Characterization of the smtA gene encoding an S-adenosylmethionine-dependent methyltransferase of Escherichia coli

Abstract The mukB operon is located at 21 min on the Escherichia coli chromosome and seems to consist of four genes, orf30 ( smtA ), mukF , mukE , and mukB . Based on sequence similarity, the promoter-proximal gene, orf30 ( smtA ), could encode an S-adenosylmethionine-dependent methyltransferase. The smtA gene is not essential for cell growth and its expression is positively regulated by H-NS, an Escherichia coli histone-like protein.     

Heparanase regulates esophageal keratinocyte differentiation through nuclear translocation and heparan sulfate cleavage

Heparanase is an endo-beta-glucuronidase that specifically cleaves heparan sulfate (HS) chains. Heparanase is involved in the process of metastasis and angiogenesis through the degradation of HS chains of the extracellular matrix and cell surface. Recently, we demonstrated that heparanase was localized in the cell nucleus of normal esophageal epithelium and esophageal cancer, and that its expression was correlated with cell differentiation. However, the nuclear function of heparanase remains unknown. To elucidate the role of heparanase in esophageal epithelial differentiation, primary human esophageal cells were grown in monolayer as well as organotypic cultures, and cell differentiation was induced. Expression of heparanase, HS, involucrin, and p27 was determined by immunostaining and Western blotting. SF4, a novel pharmacological inhibitor, was used to specifically inhibit heparanase activity. Upon esophageal cell differentiation, heparanase was translocated from the cytoplasm to the nucleus. Such translocation of heparanase appeared to be associated with the degradation of HS chains in the nucleus and changes in the expression of keratinocyte differentiation markers such as p27 and involucrin, whose induction was inhibited by SF4. Furthermore, these in vitro observations agreed with the expression pattern of heparanase, HS, involucrin, cytokeratin 13, and p27 in normal esophageal epithelium. Nuclear translocation of heparanase and its catalytic cleavage of HS may play a critical role in the differentiation of esophageal epithelial cells. Our study provides a novel insight into the role of heparanase in an essential differentiation process.