Genome-wide transcriptional orchestration of circadian rhythms in Drosophila

Circadian rhythms govern the behavior, physiology, and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both in Drosophila and in mammals. To study how gene expression is globally regulated by the clock mechanism, we used a high density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophila under light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions, and among these the expression of 115 genes was still cycling in constant darkness, i.e. under free-running conditions. Unexpectedly the expression of a large number of genes cycled exclusively under constant darkness. We found that cycling in most of these genes was lost in the arrhythmic Clock (Clk) mutant under light-dark conditions. Expression of periodically regulated genes is coordinated locally on chromosomes where small clusters of genes are regulated jointly. Our findings reveal that many genes involved in diverse functions are under circadian control and reveal the complexity of circadian gene expression in Drosophila.     

A putative met-enkephalin releaser, kyotorphin enhances intracellular Ca2+ in the synaptosomes

Kyotorphin (Tyr-Arg) at 1 to 100 microM increased the intracellular [Ca2+]i, determined with Quin-II in the slice and the entry of 45Ca2+ entry into synaptosomes of the lower brain stem of the rat. These effects were not antagonized by nifedipine nor verapamil. However, since this dipeptide caused no changes on the membrane potentials of the synaptosomes, measured with Rhodamine 6G, it is suggested that the kyotorphin-induced increase in the [Ca2+]i may be due not to effects on the voltage dependent Ca2+ channels and Na+-Ca2+ exchange mechanisms caused by the changes of the membrane potentials, but to the specific receptor (kyotorphin receptor)-mediated mechanisms.     

Detection of β-glucosidase as saprotrophic ability from an ectomycorrhizal mushroom, Tricholoma matsutake

We investigated extracellular carbohydrase production in the medium of an ectomycorrhizal fungus, Tricholoma matsutake, to reveal its ability to utilize carbohydrates such as starch as a growth substrate and to survey the saprotrophic aspects. We found β-glucosidase activity in the static culture filtrate of this fungus. The β-glucosidase was purified and characterized. The purified enzyme was obtained from about 2.1 l static culture filtrate, with 9.0% recovery, and showed a single protein band on SDS-PAGE. Molecular mass was about 160 kDa. The enzyme was most active around 60°C and pH 5.0, and stable over a pH of 4.0–8.0 for 30 min at 37°C. The purified enzyme was activated by the presence of Ca²⁺ and Mn²⁺ ions (about 2–3 times that of the control). The enzyme readily hydrolyzed oligosaccharides having a β-1,4-glucosidic linkage such as cellobiose and cellotriose. However, it did not hydrolyze polysaccharides such as avicel and CM-cellulose or oligosaccharides having an α-glucosidic linkage. Moreover, cellotriose was hydrolyzed by the enzyme for various durations, and the resultant products were analyzed by TLC. We concluded that the enzyme from T. matsutake seems to be a β-glucosidase because cellotriose with a β-1,4-glucosidic linkage decomposed to glucose during the enzyme reaction.     

Survival and death of epiblast cells during embryonic stem cell derivation revealed by long-term live-cell imaging with an Oct4 reporter system

Despite the broad literature on embryonic stem cells (ESCs), their derivation process remains enigmatic. This may be because of the lack of experimental systems that can monitor this prolonged cellular process. Here we applied a live-cell imaging technique to monitor the process of ESC derivation over 10 days from morula to outgrowth phase using an Oct4/eGFP reporter system. Our imaging reflects the ‘natural’ state of ESC derivation, as the ESCs established after the imaging were both competent in chimeric mice formation and germ-line transmission. Using this technique, ESC derivation in conventional conditions was imaged. After the blastocoel was formed, the intensity of Oct4 signals attenuated in the trophoblast cells but was maintained in the inner cell mass (ICM). Thereafter, the Oct4-positive cells scattered and their number decreased along with apoptosis of the other Oct4-nagative cells likely corresponds to trophoblast and hypoblast cells, and then only the surviving Oct4-positive cells proliferated and formed the colony. All embryos without exception passed through this cell death phase. Importantly, the addition of caspase inhibitor Z-VAD-FMK to the medium dramatically suppressed the loss of Oct4-positive cells and also other embryo-derived cells, suggesting that the cell deaths was induced by a caspase-dependent apoptotic pathway. Next we imaged the ESC derivation in 3i medium, which consists of chemical compounds that can suppress differentiation. The most significant difference between the conventional and 3i methods was that there was no obvious cell death in 3i, so that the colony formation was rapid and all of the Oct4-positive cells contributed to the formation of the outgrown colony. These data indicate that the prevention of cell death in epiblast cells is one of the important events for the successful establishment of ESCs. Thus, our imaging technique can advance the understanding of the time-dependent cellular changes during ESC derivation.     

Deficiency in Clonogenic Endometrial Mesenchymal Stem Cells in Obese Women with Reproductive Failure – a Pilot Study

Objectives

The mechanisms of obesity associated reproductive complications remain poorly understood. Endometrial mesenchymal stem-cells are critical for cyclic renewal and uterine function. Recently, W5C5⁺ cells, with high clonogenicity, capable of producing endometrial stroma in vivo, have been described. We sought to investigate the abundance and cloning efficiency of W5C5⁺ and W5C5⁻ endometrial cells in relation to Body Mass Index, age and reproductive outcome.

Design

W5C5⁺ and W5C5⁻ cells were purified from mid-luteal endometrial biopsies (n = 54) by magnetic bead separation and subjected to in vitro colony-forming assays.

Results

First trimester pregnancy losses were significantly higher in obese subjects (n = 12) compared to overweight (n = 20) and subjects with normal Body Mass Index (n = 22) (P<0.05, P<0.01, respectively). W5C5⁺ cells (%) were significantly lower in obese subjects compared to subjects with normal Body Mass Index (P<0.05). W5C5⁺ cloning efficiency was significantly lower in obese subjects compared to overweight and subjects with normal Body Mass Index (P<0.05, respectively). W5C5⁻ cloning efficiency was significantly lower in obese subjects compared to subjects with normal Body Mass Index (P<0.05). Body Mass Index was significantly negatively correlated with W5C5⁺ cloning efficiency and W5C5⁻ cloning efficiency (P<0.01, respectively), and positively correlated with first trimester loss (P<0.01). We found no significant results with age (P>0.05).

Conclusions

Our observations suggest that the regenerative capacity and plasticity of the endometrium of obese women is suboptimal, which in turn may account for the increased risk of reproductive complications associated with obesity.     

Membrane-buffer partition coefficients of tetracaine for liquid-crystal and solid-gel membranes estimated by direct ultraviolet spectrophotometry

The membrane-buffer partition coefficient of tetracaine was measured by direct ultraviolet spectrophotometry in dimyristoylphosphatidylcholine unilamellar liposomes at temperatures above and below the main phase transition. The partition coefficients of uncharged tetracaine to solid-gel (18 degrees C) and liquid-crystal (30 degrees C) membranes were 6.9 x 10(4) and 1.2 x 10(5), respectively. Despite the general assumption that local anesthetic binding to the solid membrane is negligible, this study showed that the solid membrane binding amounts to 57.5% of the liquid membrane binding. Binding of the charged form to the liquid or solid membrane was not detectable under the present experimental condition of 0.03 mM tetracaine bulk concentration. The present method measures metachromasia of local anesthetics when bound to lipid membranes. Its advantage is that the separation of the vesicles from the solution is not required. A linearized equation is presented that estimates the partition coefficient or binding constant graphically from a linear plot of the absorbance data. The method is applicable for estimation of drug partition when a measurable spectral change occurs due to complex formation.     

Characterization of two isoforms of a human DnaJ homologue,HSJ2

Two cDNA forms were characterized for a human dnaJ homologue, HSJ2. Nucleotide sequencing showed that the gene product HSJ2 was longer than previously reported, extending its homology to other human DnaJ paralogues, and that the two cDNAs encoded two proteins as a result of alternative splicing. The products were 326 amino acids (designated as HSJ2a) and 241 amino acids (HSJ2b) in length, sharing the N-terminal 231 amino acids including the DnaJ homology region. When fused to green fluorescent protein and expressed in HeLa cells, HSJ2a was found to be localized to the nucleus, indicating that HSJ2a is a nuclear co-chaperone. HSJ2b, however, was observed throughout the cell, consistent with the elimination of a putative nuclear localization signal sequence as a result of the alternative splicing.