Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells

We developed a new and efficient method for osteoblastic differentiation of human embryonic stem cells (hESCs) using primary bone-derived cells (PBDs). Three days after embryoid body (hEB) formation, cells were allowed to adhere to culture surface where PBDs were pre-plated and mitomycin C-treated in DMEM/F12 medium supplemented with 5% knockout serum replacement. As early as 14 days, mineralization and formation of nodule-like structures in cocultured hEBs were prominent by von Kossa and Alizarin S staining, and expressions of osteoblast-specific markers including bone sialoprotein, alkaline phosphates, osteocalcin, collagen 1, and core binding factor alpha1 by RT-PCR. In addition, FACS analysis revealed that over 19% of the differentiated cells expressed osteocalcin. These results suggest that PBDs not only have osteogenic effects releasing osteogenic factors as bone morphogenic protein (BMP) 2 and BMP 4 but also have exerted other effects, whether chemical or physical, for the differentiation of hESCs.     

Scanning electron microscope study of ancient parasite eggs recovered from Korean mummies of the Joseon Dynasty

We have previously shown that parasite eggs have been identified in the coprolites of Korean mummies. These eggs have shed light on parasitic infection patterns in Korean populations living several hundred years ago. We conducted a scanning electron microscopy (SEM) study on ancient Trichuris trichiura, Ascaris lumbricoides, Metagonimus yokogawai, Paragonimus westermani, and Gymnophalloides seoi eggs recovered from Korean mummies of the Joseon Dynasty. We anticipated that the taphonomic conditions of mummification would alter the eggs of certain species but not of others. Our SEM data show that each species of ancient egg exhibited different degrees of preservation. Thus, some of them, for example, M. yokogawai, exhibited a better preservation status than others, suggesting that they should be the first candidates considered when choosing subjects for future paleoparasitological studies.     

Response of diapausing eggs hatching to changes in temperature and the presence of fish kairomones

It has been hypothesized that the production of diapausing eggs in Daphnia can be induced by fish kairomones. A population of Daphnia could survive severe predation using this predator avoidance strategy. However, in changing environments, diapausing eggs experience various temperature conditions, and hatchlings at emergence may be exposed to the same predation risks as their mothers. Therefore, staying in diapause or an immediate response upon hatching to available environmental information could be important for hatchling survival. For this study, we investigated the impact of water temperature (10, 15, 20, and 25°C) in the presence and absence of fish kairomones (Lepomis macrochirus) on the hatching success of resting eggs (D. galeata). Results show that no diapausing eggs hatched at the lowest temperature (10°C), and the highest hatch percentage occurred at 15°C. Although higher water temperatures reduced hatching success, diapausing eggs hatched more quickly. The number of hatchlings was significantly higher after exposure to fish kairomones, and this was more noticeable at higher temperatures (20 and 25°C). The present results suggest that the diapausing eggs were produced as a predator avoidance strategy in Daphnia; however, the presence of fish works as a positive signal to increase hatchlings when the diapausing stage is terminated.     

Functional characterization of a bacterial expansin from Bacillus subtilis for enhanced enzymatic hydrolysis of cellulose

Expansin is a plant protein family that induces plant cell wall‐loosening and cellulose disruption without exerting cellulose‐hydrolytic activity. Expansin‐like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a β‐expansin produced by maize. Therefore, we cloned the BsEXLX1 gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a cellulose substrate, the recombinant protein exhibited both cellulose‐binding and cellulose‐weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7‐fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non‐eukaryotic source. Biotechnol. Bioeng. 2009;102: 1342–1353. © 2008 Wiley Periodicals, Inc.     

Characterization of polymorphic microsatellite loci in cultivated and wildPanax ginseng

Ginseng (Panax ginseng) is one of the most important herbal remedies used in East Asia. The present study investigated six polymorphic microsatellite markers (PG29, PG281, PG287, PG668, PG1319, and PG1481) in samples of cultivated and wildP. ginseng collected in Korea. Total allelic number observed in this study was 27 (average allelic numbers per locus: 4.5). All examined loci exhibited deviation from the Hardy-Weinberg equilibrium and deficiency of heterozygosity in both cultivated and wild groups. Although the wild ginseng group exhibited slightly more polymorphic behavior (mean PIC=0.392, GD=0.454 and H_obs=0.129), compared with the cultivated group (mean PIC=0.383, GD=0.438 and H_obs=0.105), no significant differences of allele frequencies and genotype distributions were revealed. By combined analysis of six loci in 100 cultivated ginsengs, 71 different types were observed. The analyzed microsatellite loci in this study will be helpful for understanding genetic variation, QTL mapping and phylogenic studies inPanax species.     

Synthesis and evaluation of stilbene derivatives as a potential imaging agent of amyloid plaques

Fluorescence probes that can detect Aβ (β-amyloid peptide) plaque are important tools for diagnosis of Alzheimer’s disease (AD), and 4-N-methylamino-4′-hydroxystilbene (SB-13) is one of the promising candidate molecules. We report here the synthesis of SB-13 derivatives that consist of various electron donating/withdrawing moieties and distinct size of N-substituents. The synthesized compounds were screened for detection of Aβ40 fibrils in vitro. Four compounds exhibited more than sixfold intensity increase, and they were further analyzed for detail bindings and Aβ plaque imaging. Among these molecules, compound 42 meets two critical requirements for imaging agent; high fluorescence responsiveness and strong binding affinity. This compound showed more than 25-fold increase with the dissociation constant of 1.13 ± 0.37 μM. In AD mouse brain tissue, 42 selectively stained Aβ plaque, more specifically peripheral regions of Aβ plaque. This finding demonstrated its potential use as brain-imaging agents for AD studies.     

Spatial genetic structure in populations of the terrestrial orchid Cephalanthera longibracteata (Orchidaceae)

Orchid seeds are unusual for being the smallest among flowering plants. These dust-like seeds are wind-borne and, thus, would seem to have the potential for long-distance dispersal (a common perception); this perception has led to a prediction of near-random spatial genetic structure within orchid populations. Mathematical models (e.g., simple ballistic model) for wind-dispersed seeds and wind-tunnel experiments, in contrast, indicate that most seeds of orchids should fall close to the maternal plant (<6 m), supporting a prediction of significant fine-scale genetic structure within populations. In reality we do not know much about seed dispersion in orchids. To determine which of these two predictions is more appropriate, Wright's F statistics and spatial autocorrelation analysis were used to examine the genetic structure within two adult populations of the terrestrial orchid Cephalanthera longibracteata (Orchidaceae) in southern Korea. In results comparable to those of other self-compatible, mixed-mating plant species, C. longibracteata populations exhibited low levels of genetic diversity (mean H(e) = 0.036) and a significant excess of homozygosity (mean F(IS) = 0.330), consistent with substantial inbreeding via selfing and/or mating among close relatives in a spatially structured population. Spatial autocorrelation analysis revealed significant positive genetic correlations among plants located <10 m, with relatedness at <3 m comparable to that expected for half sibs and first cousins. This genetic structure supports the prediction that the majority of seed dispersal occurs over distances of less than 10 m and is responsible for generating substantial overlap in seed shadows within C. longibracteata populations.