A globotriaosylceramide (Gb3Cer) mimic peptide isolated from phage display library expressed strong neutralization to Shiga toxins

A Gb3-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb3 antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb3 antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb3 antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb3 mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb3Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb3 mimic peptide potentially has great promise for use against Stxs.     

Interspecific variation of body shape and sexual dimorphism in three coexisting species of the genus <Emphasis Type="Italic">Petrotilapia</Emphasis> (Teleostei: Cichlidae) from Lake Malawi

Differences in color patterns have been the most used feature in describing cichlid species belonging to genus Petrotilapia from Lake Malawi. In this study, we quantified morphological variation in body shape within and among three coexisting Petrotilapia species using landmark-based geometric morphometric methods. Statistic analyses revealed significant body shape differences among species but not between sexes. Post hoc multiple comparisons based on Mahalanobis distances revealed that P. nigra was significantly different from P. genalutea and Petrotilapia sp., whereas the latter two were not significantly different. The splines generated showed that the most pronounced variation was in the head region, in which P. nigra had a relatively longer and deeper head than the other two. The most clear-cut distinction was in gape length; P. genalutea had the longest gape, followed by Petrotilapia sp., whereas P. nigra had the shortest gape. Body depth was shallower in P. nigra than the others. When comparing sexes by their centroid size, ANOVA revealed that males were bigger than females. Therefore, we conclude that color is not the only feature that can distinguish these congeners. We discuss the observed sexual dimorphism in terms of sexual selection and relate morphological variation among species to feeding behavior, which may help explain their coexistence in nature.     

Wnt proteins promote neuronal differentiation in neural stem cell culture

Wnt signaling is implicated in the control of cell growth and differentiation during CNS development from studies of mouse and chick models, but its action at the cellular level has been poorly understand. In this study, we examine the in vitro function of Wnt signaling in embryonic neural stem cells, dissociated from neurospheres derived from E11.5 mouse telencephalon. Conditioned media containing active Wnt-3a proteins are added to the neural stem cells and its effect on regeneration of neurospheres and differentiation into neuronal and glial cells was examined. Wnt-3a proteins inhibit regeneration of neurospheres, but promote differentiation into MAP2-positive neuronal cells. Wnt-3a proteins also increase the number of GFAP-positive astrocytes but suppress the number of oligodendroglial lineage cells expressing PDGFR or O4. These results indicate that Wnt-3a signaling can inhibit the maintenance of neural stem cells, but rather promote the differentiation of neural stem cells into several cell lineages.     

Surfactant-mediated gene transfer for animal cells

A commercially available cationic surfactant, dimethyl-dioctadecyl ammonium bromide (DDAB), was used for making lipid vesicles. DDAB easily dissolved in water at 60 °C and formed lipid vesicles at room temperature. The lipid vesicles showed very low cytotoxicity compared with other cationic surfactants. After the lipid vesicles were mixed with plasmid DNA solution, the solution was added to mammalian cells. The addition of a nonionic surfactant (Tween 80) to the cationic lipid vesicles at the weight ratio of 1:1 enhanced transfection efficiency. Adding more or less than the optimal amounts of DNA and lipid vesicles resulted in decreased transfection efficiency. With the optimal amounts of DNA (pCMVβ) and lipid vesicles, about 90–95% of CHO-K1 and BHK-21C13 cells transiently expressed β-galactosidase activity 24 h after transfection. By this procedure, stable transformants around 10⁵ cells corresponding to 10% efficiency could be obtained by one batch transfection. This revised version was published online in June 2006 with corrections to the Cover Date.     

Paclitaxel-induced aberrant mitosis and mitotic slippage efficiently lead to proliferative death irrespective of canonical apoptosis and p53

Spindle poisons elicit various cellular responses following metaphase arrest, but how they relate to long-term clonogenicity has remained unclear. We prepared several HeLa lines in which the canonical apoptosis pathway was attenuated, and compared their acute responses to paclitaxel, as well as long-term fate, with the parental line. Three-nanomolar paclitaxel induced brief metaphase arrest (<5 h) often followed by aberrant mitosis, and about 90% of the cells of each line had lost their clonogenicity after 48 h of the treatment. A combination of the same concentration of paclitaxel with the kinesin-5 inhibitor, S-trityl-L-cysteine (STLC), at 1 µM led to much longer arrest (～20 h) and predominance of subsequent line-specific responses: mitochondrial outer membrane permeabilization (MOMP) in the apoptosis-prone line, or mitotic slippage without obvious MOMP in the apoptosis-reluctant lines. In spite of this, combination with STLC did not lead to a marked difference in clonogenicity between the apoptosis-prone and -reluctant lines, and intriguingly resulted in slightly better clonogenicity than that of cells treated with 3 nM paclitaxel alone. This indicates that changes in the short-term response within 3 possible scenarios — acute MOMP, mitotic slippage or aberrant mitosis ― has only a weak impact on clonogenicity. Our results suggest that once cells have committed to slippage or aberrant mitosis they eventually undergo proliferative death irrespective of canonical apoptosis or p53 function. Consistent with this, cells with irregular DNA contents originating from mitotic slippage or aberrant mitosis were mostly eliminated from the population within several rounds of division after the drug treatment.     

De novo CpG methylation on an artificial chromosome-like vector maintained for a long-term in mammalian cells

Biotechnology Letters - To examine whether an autonomously replicating, artificial chromosome-like vector containing a long genomic DNA sequence (namely, Epigenosome-Nanog) undergoes de novo CpG...     

Stochastic simulation of structured skin cell population dynamics

The epidermis is the outmost skin tissue. It operates as a first defense system to process inflammatory signals and responds by producing inflammatory mediators that promote the recruitment of immune cells. Various skin diseases such as atopic dermatitis occur as a result of the defect of proper skin barrier function and successive impaired inflammatory responses. The onset of such a skin disease links to the disturbed epidermal homeostasis regulated by appropriate self-renewal and differentiation of epidermal stem cells. The theory of physiologically structured population models provides a versatile framework to formulate mathematical models which describe the growth dynamics of a cell population such as the epidermis. In this paper, we develop an algorithm to implement stochastic simulation for a class of physiologically structured population models. We demonstrate that the developed algorithm is applicable to several cell population models and typical age-structured population models. On the basis of the developed algorithm, we investigate stochastic dynamics of skin cell populations and spread of inflammation. It is revealed that demographic stochasticity can bring considerable impact on the outcome of inflammation spread at the tissue level.     

Genetic mapping of the loquat canker resistance locus in bronze loquat (Eriobotrya deflexa)

Loquat canker disease, caused by Pseudomonas syringae pv. eriobotryae, is one of the most harmful diseases of commercial cultivars of loquat (Eriobotrya japonica). To introgress resistance to loquat canker, we identified the linkage group and position of the resistance locus derived from the related wild species bronze loquat (Eriobotrya deflexa). The segregation of resistance and susceptibility fit the expected ratio of 1:1 in 96 individuals from a three-way cross involving bronze loquat (heterozygous for resistance) and two cultivars of loquat (susceptible). The genomic region containing Pse-a was identified by using a genome scanning approach, and the loquat canker resistance locus was mapped at the top of linkage group 10 by applying novel simple sequence repeat (SSR) markers designed on the basis of the ‘Golden Delicious’ apple genome sequence. The constructed linkage group spans 69.4 cM and has an average marker density of 2.6 cM per marker. The developed molecular markers tightly linked to the loquat canker resistance locus will be useful for marker-assisted selection and for introgression of resistance into loquat in breeding programs.     

Evaluation of extensional and torsional stiffness of single actin filaments by molecular dynamics analysis

It is essential to investigate the mechanical behaviour of cytoskeletal actin filaments in order to understand their critical role as mechanical components in various cellular functional activities. These actin filaments consisting of monomeric molecules function in the thermal fluctuations. Hence, it is important to understand their mechanical behaviour on the microscopic scale by comparing the stiffness based on thermal fluctuations with the one experimentally measured on the macroscopic scale. In this study, we perform a large-scale molecular dynamics (MD) simulation for a half-turn structure of an actin filament. We analyse its longitudinal and twisting Brownian motions in equilibrium and evaluated its apparent extensional and torsional stiffness on the nanosecond scale. Upon increasing the sampling-window durations for analysis, the apparent stiffness gradually decreases and exhibits a trend to converge to a value that is close to the experimental value. This suggests that by extrapolating the data obtained in the MD analysis, we can estimate the experimentally determined stiffness on the microsecond to millisecond scales. For shorter temporal scales, the apparent stiffness is larger than experimental values, indicating that fast, local motions of the molecular structure are dominant. To quantify the local structural changes within the filament on the nanosecond scale and investigate the molecular mechanisms, such as the binding of the actin-regulatory proteins to the filaments, it is preferable to analyse the mechanical behaviour on the nanometre and nanosecond scales using MD simulation.