Type IV pilin is glycosylated in Pseudomonas syringae pv. tabaci 6605 and is required for surface motility and virulence

Type IV pilin (PilA) is a major constituent of pilus and is required for bacterial biofilm formation, surface motility and virulence. It is known that mature PilA is produced by cleavage of the short leader sequence of the pilin precursor, followed by methylation of N-terminal phenylalanine. The molecular mass of the PilA mature protein from the tobacco bacterial pathogen Pseudomonas syringae pv. tabaci 6605 (Pta 6605) has been predicted to be 12 329 Da from its deduced amino acid sequence. Previously, we have detected PilA as an approximately 13-kDa protein by immunoblot analysis with anti-PilA-specific antibody. In addition, we found the putative oligosaccharide-transferase gene tfpO downstream of pilA. These findings suggest that PilA in Pta 6605 is glycosylated. The defective mutant of tfpO (ΔtfpO) shows reductions in pilin molecular mass, surface motility and virulence towards host tobacco plants. Thus, pilin glycan plays important roles in bacterial motility and virulence. The genetic region around pilA was compared among P. syringae pathovars. The tfpO gene exists in some strains of pathovars tabaci, syringae, lachrymans, mori, actinidiae, maculicola and P. savastanoi pv. savastanoi. However, some strains of pathovars tabaci, syringae, glycinea, tomato, aesculi and oryzae do not possess tfpO, and the existence of tfpO is independent of the classification of pathovars/strains in P. syringae. Interestingly, the PilA amino acid sequences in tfpO-possessing strains show higher homology with each other than with tfpO-nonpossessing strains. These results suggest that tfpO and pilA might co-evolve in certain specific bacterial strains.     

Virulence factor regulator (Vfr) controls virulence‐associated phenotypes in Pseudomonas syringae pv. tabaci 6605 by a quorum sensing‐independent mechanism

Virulence factor regulator (Vfr) is a member of the cyclic 3′,5′‐adenosine monophosphate (cAMP) receptor proteins that regulate the expression of many important virulence genes in Pseudomonas aeruginosa. The role of Vfr in pathogenicity has not been elucidated fully in phytopathogenic bacteria. To investigate the function of Vfr in Pseudomonas syringae pv. tabaci 6605, the vfr gene was disrupted. The virulence of the vfr mutant towards host tobacco plants was attenuated significantly, and the intracellular cAMP level was decreased. The vfr mutant reduced the expression of flagella‐, pili‐ and type III secretion system‐related genes and the defence response in nonhost Arabidopsis leaves. Furthermore, the expression levels of achromobactin‐related genes and the iron uptake ability were decreased, suggesting that Vfr regulates positively these virulence‐related genes. In contrast, the vfr mutant showed higher tolerance to antimicrobial compounds as a result of the enhanced expression of the resistance–nodulation–division family members, the mexA, mexB and oprM genes. We further demonstrated that the mutant strains of vfr and cyaA, an adenylate cyclase gene responsible for cAMP synthesis, showed a similar phenotype, suggesting that Vfr regulates virulence factors in a cAMP‐dependent manner. Because there was no significant difference in the production of acylhomoserine lactone (AHL) quorum sensing molecules in the wild‐type, vfr and cyaA mutant strains, Vfr might control important virulence factors by an AHL‐independent mechanism in an early stage of infection by this bacterium.     

A fatty acid synthase blockade induces tumor cell-cycle arrest by down-regulating Skp2

In eukaryotes, fatty acid synthase (FAS) is the enzyme responsible for synthesis of palmitate, the precursor of long-chain nonessential fatty acids. FAS is up-regulated in a wide range of cancers and has been suggested as a relevant drug target. Here, two independent approaches are taken toward knocking down FAS and then probing its connection to tumor cell proliferation. In one approach, Orlistat, a drug approved for treating obesity, is used as a potent inhibitor of the thioesterase function of FAS. In a separate strategy, the expression of FAS is suppressed by targeted knock-down with small interfering RNA. In both circumstances, the ablation of FAS activity causes a dramatic down-regulation of Skp2, a component of the E3 ubiquitin ligase that controls the turnover of p27Kip1. These effects ultimately tie into the retinoblastoma protein pathway and lead to a cell-cycle arrest at the G1/S boundary. Altogether, the findings of the study reveal unappreciated links between fatty acid synthase and ubiquitin-dependent proteolysis of cell-cycle regulatory proteins.     

Effects of Ultraviolet Light on Melanocyte Differentiation: Studies with Mouse Neural Crest Cells and Neural Crest‐derived Cell Lines

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L ‐3,4‐dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT‐ or DOPA‐positive cells between the UV‐irradiated cultures and the non‐irradiated cultures. We then examined the effects of UV light on KIT‐positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase‐positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with α‐melanocyte‐stimulating hormone (α‐MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase‐negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal‐regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as α‐MSH and/or endothelin‐1.     

DA-Raf1, a competent intrinsic dominant-negative antagonist of the Ras-ERK pathway, is required for myogenic differentiation

Ras activates Raf, leading to the extracellular-regulated kinase (ERK)-mitogen-activated protein kinase pathway, which is involved in a variety of cellular, physiological, and pathological responses. Thus, regulators of this Ras-Raf interaction play crucial roles in these responses. In this study, we report a novel regulator of the Ras-Raf interaction named DA-Raf1. DA-Raf1 is a splicing isoform of A-Raf with a wider tissue distribution than A-Raf. It contains the Ras-binding domain but lacks the kinase domain, which is responsible for activation of the ERK pathway. As inferred from its structure, DA-Raf1 bound to activated Ras as well as M-Ras and interfered with the ERK pathway. The Ras-ERK pathway is essential for the negative regulation of myogenic differentiation induced by growth factors. DA-Raf1 served as a positive regulator of myogenic differentiation by inducing cell cycle arrest, the expression of myogenin and other muscle-specific proteins, and myotube formation. These results imply that DA-Raf1 is the first identified competent, intrinsic, dominant-negative antagonist of the Ras-ERK pathway.     

Comparison of effects of spatial autocorrelation on distribution predictions of four rare plant species in the Watarase wetland

We tested the effectiveness of distribution-prediction models for four rare herbaceous wetland species in the Watarase wetland, Japan, based on data obtained from aerial images. We used visible and near-infrared aerial images from three seasons, and elevations and vegetation heights derived from the images. Because spatial autocorrelation in species distribution data often biases the estimated effects of certain variables and reduces the prediction accuracy of distribution models, we compared the predictions of an intrinsic conditional autoregressive (CAR) model, which accounts for spatial autocorrelation, with those of a standard logistic regression model. The four study species had different distribution patterns: Ophioglossum namegatae and Impatiens ohwadae had aggregated distributions, whereas Galium tokyoense and Thalictrum simplex var. brevipes had scattered distributions. Predictions based on remote sensing images performed well for O. namegatae with the intrinsic CAR model and for I. ohwadae with both the logistic and CAR models; performance was poor for G. tokyoense and T. simplex var. brevipes with both models. Prediction accuracy improved by the CAR model in comparison to the logistic model most in O. namegatae and least in I. ohwadae. Impatiens ohwadae’s distribution was explained well by ground height. In contrast, the apparent improvement in the prediction for O. namegatae resulted from a substantial spatial random effect, suggesting the presence of determinants that could not be detected by remote sensing. The number of explanatory variables with large effects decreased in the intrinsic CAR model in three species possibly by avoiding spatial pseudoreplication, but not for T. simplex var. brevipes.     

PtdIns(3,4,5)P3 regulates spindle orientation in adherent cells

Cultured adherent cells divide on the substratum, leading to formation of the cell monolayer. However, how the orientation of this anchorage-dependent cell division is regulated remains unknown. We have previously shown that integrin-dependent adhesion orients the spindle parallel to the substratum, which ensures this anchorage-dependent cell division. Here, we show that phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is essential for this spindle orientation control. In metaphase, PtdIns(3,4,5)P3 is accumulated in the midcortex in an integrin-dependent manner. Inhibition of phosphatidylinositol-3-OH kinase (PI(3)K) reduces the accumulation of PtdIns(3,4,5)P3 and induces spindle misorientation. Introduction of PtdIns(3,4,5)P3 to these cells restores the midcortical accumulation of PtdIns(3,4,5)P3 and proper spindle orientation. PI(3)K inhibition causes dynein-dependent spindle rotations along the z-axis, resulting in spindle misorientation. Moreover, dynactin, a dynein-binding partner, is accumulated in the midcortex in a PtdIns(3,4,5)P3-dependent manner. We propose that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on spindles to the midcortex, and thereby orients the spindle parallel to the substratum.     

Oviposition-stimulatory activity of phenanthroindolizidine alkaloids of host-plant origin to a danaid butterfly, Ideopsis similis

Oviposition response of Ideopsis similis (L.) (Lepidoptera: Danaidae) was examined for 12 phenanthroindolizidine alkaloids present in its host plant, Tylophora tanakae (Maxim.) (Asclepiadaceae). At least five alkaloids, i.e. (+)‐isotylocrebrine (3,4,6,7‐tetramethoxyphenanthroindolizidine; l ), (+)‐3‐demethyliso‐ tylocrebrine ( 3 ), (+)‐isotylocrebrine N‐oxide ( 5 ), (+)‐6‐demethyltylocrebrine ( 8 ) and (–)‐7‐demethyltylophorine ( 10 ), were found to individually stimulate oviposition by females. Of these, compounds 1, 3 and 10 were regarded as key components most responsible for host recognition or preference. However, female egg‐laying was much higher in response to a mixture of the five alkaloids. In two‐choice bioassays, more eggs were deposited on samples comprising the five alkaloids than on samples consisting of a single alkaloid. This suggests strongly that host selection by the butterfly is mediated by the synergistic action of several phenanthroindolizidine alkaloids present in the host plant.     

Inbred strains of brine shrimp derived from Artemia franciscana: lineage, RAPD analysis, life span, reproductive traits and mode, adaptation, and tolerance to salinity changes

Inbred strains of the brine shrimp were developed from dry dormant cysts of wild-type Artemia franciscana produced in the Great Salt Lake, U.S.A. The established strains were named GSL2, 4, and 7. They were raised in 2% natural sea salt solution at 28 degrees C under a long-day condition, and fed on food sold for Artemia. Ovoviviparous offspring (free-swimming nauplii) in each brood derived from full sib (sister x brother) matings were used for succeeding generations. The ordinal number of the filial generation increased at a rate of ten generations per year. The number was over 60, and the lineage was recorded. Random amplified polymorphic DNA (RAPD) analyses of the inbred strains revealed the uniqueness, homogeneity, and genetic similarity among them. Their life span, the time required to become sexually mature, brood size, mode of reproduction, and adaptation and tolerance to salinity changes were investigated. The inbred strains usually released free-swimming nauplii rather than spawning encysted gastrulae (dormant cysts). On the other hand, the opposite results were obtained from wild-type Artemia under the same conditions. Both adults and nauplii of the inbred strains appeared to be less adaptive and less tolerant to salinity changes compared to those of the wild type. The established inbred strains should provide a wider and deeper scope for Artemia biology in particular, and the life sciences in general.