Calreticulin affects fibronectin-based cell-substratum adhesion via the regulation of c-Src activity

Calreticulin is an endoplasmic reticulum Ca2+-storage protein, which influences gene expression and cell adhesion. In this study, we show that calreticulin induces fibronectin gene expression and matrix deposition, leading to differences in cell spreading and focal adhesion formation in cells differentially expressing calreticulin. We further show that these effects of calreticulin occur via a c-Src-regulated pathway and that c-Src activity is inversely related to calreticulin abundance. Since c-Src is an important regulator of focal contact turnover, we investigated the effect of c-Src inhibition on cells differentially expressing calreticulin. Inhibition of c-Src rescued the poorly adhesive phenotype of the calreticulin-underexpressing cells in that they became well spread, commenced formation of numerous focal contacts, and deposited a rich fibronectin matrix. Importantly, we show that c-Src activity is dependent on releasable Ca2+ from the endoplasmic reticulum, thus implicating Ca2+-sensitive pathways that are affected by calreticulin in cell-substratum adhesion. We propose that calreticulin affects fibronectin synthesis and matrix assembly via the regulation of fibronectin gene expression. In parallel, calcium-dependent effects of calreticulin on c-Src activity influence the formation and/or stability of focal contacts, which are instrumental in matrix assembly and remodeling.     

GAzer: gene set analyzer

Gene Set Analyzer (GAzer) is a web-based integrated gene set analysis tool covering previously reported parametric and non-parametric models. Based on a simulation test for the reported algorithms, we classified and implemented three main statistical methods consisting of the z-statistic, gene permutation and sample permutation for ten gene set categories including Gene Ontology (GO) for human, mouse, rat and yeast. This tool identifies significantly altered gene sets scored by z-statistics and P-values from the z-test or permutation test and provides q-values and Bonferroni P-values to correct multiple hypothesis testing. GAzer allows users to observe changes in expression of each gene in a gene set or to see the significance of the gene sets containing a gene(s) of interest, thus allowing interactive data analysis both at the gene and gene set level. Moreover, GAzer offers extensive annotation for each gene. AVAILABILITY: The GAzer gene set analyzer is freely available at http://integromics.kobic.re.kr/GAzer/. SUPPLEMENTARY INFORMATION: This can be found on the web page (http://integromics.kobic.re.kr/GAzer/supplement.jsp).     

Cell elongation in Arabidopsis hypocotyls involves dynamic changes in cell wall thickness

Field-emission scanning electron microscopy was used to measure wall thicknesses of different cell types in freeze-fractured hypocotyls of Arabidopsis thaliana. Measurements of uronic acid content, wall mass, and wall volume suggest that cell wall biosynthesis in this organ does not always keep pace with, and is not always tightly coupled to, elongation. In light-grown hypocotyls, walls thicken, maintain a constant thickness, or become thinner during elongation, depending upon the cell type and the stage of growth. In light-grown hypocotyls, exogenous gibberellic acid represses the extent of thickening and promotes cell elongation by both wall thinning and increased anisotropy during the early stages of hypocotyl elongation, and by increased wall deposition in the latter stages. Dark-grown hypocotyls, in the 48 h period between cold imbibition and seedling emergence, deposit very thick walls that subsequently thin in a narrow developmental window as the hypocotyl rapidly elongates. The rate of wall deposition is then maintained and keeps pace with cell elongation. The outer epidermal wall is always the thickest ( approximately 1 mum) whereas the thinnest walls, about 50 nm, are found in inner cell layers. It is concluded that control of wall thickness in different cell types is tightly regulated during hypocotyl development, and that wall deposition and cell elongation are not invariably coupled.     

Genetic diversity in laboratory colonies of western corn rootworm (Coleoptera: Chrysomelidae), including a nondiapause colony

Laboratory-reared western corn rootworms, Diabrotica virgifera virgifera, from colonies maintained at the North Central Agricultural Research Laboratory (NCARL) in Brookings, SD, are used extensively by many researchers in studies of the biology, ecology, behavior, and genetics of this major insect pest. A nondiapause colony developed through artificial selection in the early 1970s is particularly attractive for many studies because its generation time is much shorter than that of typical diapause colonies. However, the nondiapause colony has been in culture for approximately 190 generations without out-crossing. We compared variation at six microsatellite loci among individuals from the NCARL nondiapause colony (approximately 190 generations), main diapause colony (approximately 22 generations), four regional diapause colonies (3-8 generations), and four wild populations. Genetic diversity was very similar among the diapause laboratory colonies and wild populations. However, the nondiapause colony showed approximately 15-39% loss of diversity depending on the measure. Pairwise estimates of F(ST) were very low, revealing little genetic differentiation among laboratory colonies and natural populations. The nondiapause colony showed the greatest genetic differentiation with an average pairwise F(ST) of 0.153. There was little evidence that the laboratory colonies had undergone genetic bottlenecks except for the nondiapause colony. The nondiapause colony has suffered a moderate loss in genetic diversity and is somewhat differentiated from wild populations. This was not unexpected given its history of artificial selection for the nondiapause trait, and the large number of generations in culture. In contrast, the results indicate that the diapause colonies maintained at NCARL are genetically similar to wild populations.     

Effects of daunorubicin on ganglioside expression and neuronal differentiation of mouse embryonic stem cells

Gangliosides are implicated in neuronal development processes. The regulation of ganglioside levels is closely related to the induction of neuronal cell differentiation. In this study, the relationship between ganglioside expression and neuronal cell development was investigated using an in vitro model of neural differentiation from mouse embryonic stem (mES) cells. Daunorubicin (DNR) was applied to induce the expression of gangliosides in embryoid body (EB) (4+). We observed an increase in expression of gangliosides in all stages of EBs by treatment of DNR (2microM). High-performance thin-layer chromatography (HPTLC) showed that gangliosides GD3, GD1a, GT1b, and GQ1b increased in DNR-treated 7-day-old EB (4+) [EB (4+):7]. DNR treatment significantly increased the expression of gangliosides, especially GT1b and GQ1b in comparison to control cells. Interestingly, GQ1b co-localized with microtubule-associated protein 2 (MAP-2) expressing cells in DNR-treated EB (4+):7. The co-localization of GQ1b and MAP-2 was found in neurite-bearing cells in DNR-treated 15-day-old EB (4+) [EB (4+):15], whereas no significant expression of GQ1b and less neurite formation were observed in untreated control. Also, the expression of synaptophysin and NF200, both neuronal markers associated with neruites, was increased by DNR treatment. These results demonstrate that DNR increases expression of gangliosides, especially GQ1b, in differentiating neuronal cells. Further, neurite-bearing neuronal cell differentiation can be facilitated by DNR, possibly through the induction of gangliosides. Thus, the present data suggest that DNR is beneficial for facilitating neuronal differentiation from ES cells and among the gangliosides analyzed in the present study, GQ1b is mainly involved in neurite formation.     

Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells

Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation.     

Complete nucleotide sequence and organization of the mitogenome of the silk moth Caligula boisduvalii (Lepidoptera: Saturniidae) and comparison with other lepidopteran insects

The 15,360-bp long complete mitogenome of Caligula boisduvalii possesses a gene arrangement and content identical to other completely sequenced lepidopteran mitogenomes, but different from the common arrangement found in most insect order, as the result of the movement of tRNA(Met) to a position 5'-upstream of tRNA Ile. The 330-bp A+T-rich region is apparently capable of forming a stem-and-loop structure, which harbors the conserved flanking sequences at both ends. Dissimilar to what has been seen in other sequenced lepidopteran insects, the initiation codon for C. boisduvalii COI appears to be TTG, which is a rare, but apparently possible initiation codon. The ATP8, ATP6, ND4L, and ND6 genes, which neighbor another PCG at their 3' end, all harbored potential sequences for the formation of a hairpin structure. This is suggestive of the importance of such structures for the precise cleavage of the mRNA of mature PCGs. Phylogenetic analyses of available sequenced species of Bombycoidea, Pyraloidea, and Tortricidea supported the morphology-based current hypothesis that Bombycoidea and Pyraloidea are monophyletic (Obtectomera). As previously suggested, Bombycidae (Bombyx mori and B. mandarina) and Saturniidae (Antheraea pernyi and C. boisduvalii) formed a reciprocal monophyletic group.     

Pseudomonas fluorescens HYK0210-SK09 offers species-specific biological control of winter algal blooms caused by freshwater diatom Stephanodiscus hantzschii

Aims:  The study of an algicidal activity and mechanism of the isolated Pseudomonas fluorescens HYK0210-SK09 (SK09) against a winter bloomed harmful diatom, Stephanodiscus hantzschii.
Methods and Results:  SK09 was isolated from the Paldang reservoir, Korea and used to biological control of S. hantzschii . The inoculation of SK09 at the final density of 5 × 10⁶ cells ml⁻¹ caused degradation of >90% of S. hantzschii cells within 5 days. The algal cell lysis was achieved by a direct attack of the bacteria to the diatom cells, and the algicidal compound was located in the cytoplasm of the cell. As SK09 did not suppress Microcystis aeruginosa , Anabaena cylindrica , Coelastrum astroideum or Cyclotella meneghiniana , it appeared to attack S. hantzschii in a species-specific manner. Testing in an indoor mesocosms confirmed that SK09 effectively reduced S. hantzschii cells by 88% within 9 days.
Conclusions:  This bacterium is useful in regulating blooms of S. hantzschii . However, it should be studied in the future that their impact in shaping phytoplankton community and their activity in natural environments.
Significance and Impact of the Study:  The bacterium enabled us to develop a new strategy, to understand the interaction for anthropogenic control of harmful algal blooms in nature.     

Gene expression profiling demonstrates a novel role for foetal fibrocytes and the umbilical vessels in human fetoplacental development

There is a difference in the susceptibility to inflammation between the umbilical vein (UV) and the umbilical arteries (UAs). This led us to hypothesize that there is an intrinsic difference in the pro-inflammatory response between UA and UV. Real-time quantitative RT-PCR and microarray analysis revealed higher expression of interleukin (IL)-1β and IL-8 mRNA in the UV and differential expression of 567 genes between the UA and UV associated with distinct biological processes, including the immune response. Differential expression of human leukocyte antigen (HLA)-DRA mRNA between the UA and UV was due to unexpected HLA-DR⁺ cells migrating via the umbilical vessels into Wharton's jelly, more frequently in the UV. A significant proportion of these cells co-expressed CD45 and type I pro-collagen, and acquired CD163 or α-smooth muscle actin immunoreactivity in Wharton's jelly. Migrating cells were also found in the chorionic and stem villous vessels. Furthermore, the extent of migration increased with progression of gestation, but diminished in intrauterine growth restriction (IUGR). The observations herein strongly suggest that circulating foetal fibrocytes, routing via umbilical and placental vessels, are a reservoir for key cellular subsets in the placenta. This study reports fibrocytes in the human umbilical cord and placenta for the first time, and a novel role for both circulating foetal cells and the umbilical vessels in placental development, which is deranged in IUGR.