Induction of lung-like cells from mouse embryonic stem cells by decellularized lung matrix

Decellularization of tissues is a recently developed technique mostly used to provide a 3-dimensional matrix structure of the original organ, including decellularized lung tissues for lung transplantation. Based on the results of the present study, we propose new utilization of decellularized tissues as inducers of stem cell differentiation. Decellularized lung matrix (L-Mat) samples were prepared from mouse lungs by SDS treatment, then the effects of L-Mat on differentiation of ES cells into lung cells were investigated. ES cell derived-embryoid bodies (EBs) were transplanted into L-Mat samples and cultured for 2 weeks. At the end of the culture, expressions of lung cell-related markers, such as TTF-1 and SP-C (alveolar type II cells), AQP5 (alveolar type I cells), and CC10 (club cells), were detected in EB outgrowths in L-Mat, while those were not found in EB outgrowths attached to the dish. Our results demonstrated that L-Mat has an ability to induce differentiation of ES cells into lung-like cells.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10^?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10^?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-¹⁴C revealed that l-glutamic acid-1-¹⁴C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme.     

Delayed sleep/wake rhythm and excessive daytime sleepiness correlate with decreased daytime brain activity during cognitive task in university students

Insufficient sleep and irregular sleep/wake rhythm are common problems among university students. We investigated the effect of sleep/wake rhythm and excessive daytime sleepiness (EDS) on the cortical oxygenation as measured by near-infrared spectroscopy (NIRS) and cognitive performance in university students. Peak- and integral values by a word fluency task were measured with NIRS. EDS was evaluated by the Epworth sleepiness scale (ESS), and performance function was evaluated using N-back task. Peak cerebral oxygenation was significantly correlated with ESS, bedtime, wake-up time, and median time of sleep. Accuracy on 2-back task was significantly correlated with integral value. Peak- and integral values were significantly lower, and bedtime and median time of sleep were significantly delayed in the EDS group than in the non-EDS group. EDS accompanied by delayed sleep/wake rhythm and short sleep duration may play an important role in decreasing daytime brain activity and cognitive performance.     

Maximum Likelihood Analysis of the Complete Mitochondrial Genomes of Eutherians and a Reevaluation of the Phylogeny of Bats and Insectivores

The complete mitochondrial genomes of two microbats, the horseshoe bat Rhinolophus pumilus, and the Japanese pipistrelle Pipistrellus abramus, and that of an insectivore, the long-clawed shrew Sorex unguiculatus, were sequenced and analyzed phylogenetically by a maximum likelihood method in an effort to enhance our understanding of mammalian evolution. Our analysis suggested that (1) a sister relationship exists between moles and shrews, which form an eulipotyphlan clade; (2) chiropterans have a sister-relationship with eulipotyphlans; and (3) the Eulipotyphla/Chiroptera clade is closely related to fereuungulates (Cetartiodactyla, Perissodactyla and Carnivora). Divergence times on the mammalian tree were estimated from consideration of a relaxed molecular clock, the amino acid sequences of 12 concatenated mitochondrial proteins and multiple reference criteria. Moles and shrews were estimated to have diverged approximately 48 MyrBP, and bats and eulipotyphlans to have diverged 68 MyrBP. Recent phylogenetic controversy over the polyphyly of microbats, the monophyly of rodents, and the position of hedgehogs is also examined. Received: 21 December 2000 / Accepted: 16 February 2001     

Cyanobacterial Cell Lineage Analysis of the Spatiotemporal hetR Expression Profile during Heterocyst Pattern Formation in Anabaena sp. PCC 7120

Diazotrophic heterocyst formation in the filamentous cyanobacterium, Anabaena sp. PCC 7120, is one of the simplest pattern formations known to occur in cell differentiation. Most previous studies on heterocyst patterning were based on statistical analysis using cells collected or observed at different times from a liquid culture, which would mask stochastic fluctuations affecting the process of pattern formation dynamics in a single bacterial filament. In order to analyze the spatiotemporal dynamics of heterocyst formation at the single filament level, here we developed a culture system to monitor simultaneously bacterial development, gene expression, and phycobilisome fluorescence. We also developed micro-liquid chamber arrays to analyze multiple Anabaena filaments at the same time. Cell lineage analyses demonstrated that the initial distributions of hetR::gfp and phycobilisome fluorescence signals at nitrogen step-down were not correlated with the resulting distribution of developed heterocysts. Time-lapse observations also revealed a dynamic hetR expression profile at the single-filament level, including transient upregulation accompanying cell division, which did not always lead to heterocyst development. In addition, some cells differentiated into heterocysts without cell division after nitrogen step-down, suggesting that cell division in the mother cells is not an essential requirement for heterocyst differentiation.     

Antimicrobial substances from rhizomes of the giant knotweed Polygonum sachalinense against the fish pathogen Photobacterium damselae subsp. piscicida

The antimicrobial compounds against the fish pathogen Photobacterium damselae subsp. piscicida were isolated from Polygonum sachalinense rhizomes. The structures of the antimicrobial compounds 1 and 2 were determined by 1H and 13C NMR, 2D-NMR (COSY, HSQC, HMBC and ROESY) and FAB-MS to be phenylpropanoid glycosides, vanicoside A and B, respectively. Both compounds have feruloyl and p-coumaroyl groups bonded to a sucrose moiety in their structures. Vanicoside A also has an acetyl group in the sucrose moiety. The MIC values for vanicoside A and B against Ph. damselae subsp. piscicida DPp-1 were 32 and 64 microg/ml, respectively. The antimicrobial activities of these vanicosides were modest, in contrast to higher activities (MICs at < 4 microg/ml) of antibiotics, florphenicol, ampicillin and amoxicillin, which have been generally used for treating pasteurellosis. The activities of the vanicosides, however, were higher than those (MICs at 256 microg/ml) of ferulic acid and p-coumaric acid. It was suggested that the structure of phenylpropanoids esterified with sucrose was essential for higher antimicrobial activity of vanicosides and also acetylation of sucrose might affect the activity against the bacterium.     

Preferential increase in the hippocampal synaptic vesicle protein 2A (SV2A) by pentylenetetrazole kindling

The present study evaluated the expressional levels of synaptic vesicle protein 2A (SV2A) and other secretary machinery proteins (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, Munc18-1, N-ethylmaleimide-sensitive factor (NSF) and soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)) in a pentylenetetrazole (PTZ) kindling model. Repeated administration of sub-convulsive PTZ (40 mg/kg, i.p.) progressively increased seizure susceptibility in mice and consistently induced clonic seizures in most animals tested at 15 days after the treatment. Western blot analysis revealed that, among the secretary machinery proteins examined, hippocampal SV2A was selectively elevated by PTZ kindling. PTZ kindling-induced SV2A expression appeared region-specific and the SV2A levels in the cerebral cortex or cerebellum were unaltered. In addition, SV2A expression by PTZ kindling was prominent in the hilar region of the dentate gyrus (DG) where GABAergic interneurons are located, but not in other hippocampal regions (e.g., the stratum lucidum of the CA3 and synaptic layers surrounding CA1 or CA3 pyramidal neurons). These findings suggest that PTZ kindling preferentially elevates SV2A expression in the hippocampus probably as a compensatory mechanism to activate the inhibitory neurotransmission.     

8-Hydroxyguanine levels and repair capacity during mouse embryonic stem cell differentiation

To evaluate the defence capacities of embryonic stem (ES) cells against gene impairment, this study measured the levels of 8-hydroxyguanine (8-OH-Gua), a well-known marker of oxidative stress in DNA, and its repair capacity during differentiation. Undifferentiated ES cells (EB3) were cultured without leukaemia inhibitory factor (LIF) for 0, 4 and 7 days and are referred to as ES-D0, ES-D4 and ES-D7, respectively. These three cell lines were treated with 300 μM hydrogen peroxide (H(2)O(2)) for 48 and 72 h. After treatment, the amounts of 8-OH-Gua in the cells were determined by the high-performance liquid chromatography (HPLC)-electrochemical detector (ECD) method. The levels of 8-OH-Gua in ES-D7 treated with H(2)O(2) were higher than those in ES-D0 and ES-D4, suggesting that the DNA in the undifferentiated cells was protected against gene impairment, as compared to that in the differentiated cells. To examine the repair capacity for 8-OH-Gua, this study analysed the expression of 8-OH-Gua repair-associated genes, 8-oxoguanine DNA glycosylase 1 (OGG1), MutY homolog (MUTYH) and Mut T homolog 1 (MTH1), in ES-D0, ES-D4 and ES-D7. The mRNA levels of MUTYH and MTH1 showed no significant change, whereas OGG1 mRNA was significantly decreased in ES-D7 treated with H(2)O(2). Moreover, it was observed that ES-D7 treated with H(2)O(2) readily underwent apoptosis, in comparison to its undifferentiated counterparts, ES-D0 and ES-D4. Taken together, ES cells are more resistant to DNA oxidative stresses than differentiated cells.