To achieve an integrated understanding of the stem cell system of planarians at both the cellular and molecular levels, we developed a new method by combining ''fluorescent activated cell sorting (FACS) index sorting'' analysis and single-cell reverse transcription-polymerase chain reaction (RT-PCR) to detect the gene expression and cell cycle state of stem cells simultaneously. Single cells were collected using FACS, and cDNAs of each cell were used for semi-quantitative RT-PCR. The results were plotted on the FACS sorting profile using the ''index sorting'' function, which enabled us to analyze the gene expression in combination with cell biological data (such as cell cycle phase) for each cell. Here we investigated the adult stem cells of planarians using this method and obtained findings suggesting that the stem cells might undergo commitment during S to G2 ⁄ M phase. This method could be a powerful and straightforward tool for examining the stem cell biology of not only planarians but also other organisms, including vertebrates.

Planarians are attractive animals in which various questions related to the central nervous system (CNS) can be addressed, such as its origin and evolution, its degree of functional conservation among different organisms, and the plasticity and regenerative capabilities of neural cells and networks. However, it is first necessary to characterize at the gene expression level how this CNS is organized in intact animals. Previous studies have shown that the planarian brain can be divided into at least three distinct domains based on the expression of otd/Otx-related genes. In order to further characterize the planarian brain, we have recently isolated a large number of planarian neural-specific genes through DNA microarrays and ESTs projects. Here, we describe new molecular domains within the brain of intact planarians by the expression of 16 planarian neural-specific genes, including the putative homologues of protein tyrosine phosphatase receptor, synaptotagmin VII, slit, G protein and glutamate and acetylcholine receptors, by in situ hybridization in both whole-mount and transverse sections. Our results indicate that planarian otd/Otx-positive domains can be further subdivided into distinct molecular regions according to the expression of different neural genes. We found differences at the gene expression level between the dorsal and ventral sides of the brain, along its antero-posterior axis and also between the proximal and distal parts of the brain lateral branches. This high level of regionalization in the planarian brain contrasts with its apparent simplicity at the morphological level.

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A novel NADH-dependent glyoxylate reductase has been found in a hyperthermophilic archaeon Thermococcus litoralis DSM 5473. This is the first evidence for glyoxylate metabolism and its corresponding enzyme in hyperthermophilic archaea. NADH-dependent glyoxylate reductase was purified < 560-fold from a crude extract of the hyperthermophile by five successive column chromatographies and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 76 kDa, and the enzyme consisted of a homodimer with a subunit molecular mass of < 37 kDa. The optimum pH and temperature for enzyme activity were < 6.5 and 90 8C, respectively. The enzyme was extremely thermostable; the activity was stable up to 90 8C. The glyoxylate reductase catalyzed the reduction of glyoxylate and hydroxypyruvate, and the relative activity for hydroxypyruvate was < one-quarter that of glyoxylate in the presence of NADH as an electron donor. NADPH exhibited rather low activity as an electron donor compared with NADH. The K m values for glyoxylate, hydroxypyruvate, and NADH were determined to be 0.73, 1.3 and 0.067 mM, respectively. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the glyoxylate reductase gene was determined and found to encode a peptide of 331 amino acids with a calculated relative molecular mass of 36 807. The amino-acid sequence of the T. litoralis enzyme showed high similarity with those of probable dehydrogenases in Pyrococcus horikoshii and P. abyssi. The purification of the enzyme from recombinant E. coli was much simpler compared with that from T. litoralis; only two steps of heat treatment and dyeaffinity chromatography were needed.Keywords: archaeal glyoxylate metabolism; gene cloning and sequencing; hyperthermophilic archaeon; NADHglyoxylate reductase; Thermococcus litoralis.Thermococcus litoralis is a typical marine hyperthermophilic archaeon that can grow near the boiling temperature of water like Pyrococcus furiosus [1]. T. litoralis is known to utilize anaerobically maltose and cellobiose as the carbon and energy sources in a modified Embden -Meyerhof pathway similar to that of many other Thermococcus and Pyrococcus species [2,3]. The modified Embden -Meyerhof pathway contains several unique enzymes such as ADP-dependent glucokinase [4,5] and ADP-dependent phosphofructokinase [6], the ferredoxin-dependent glyceraldehyde-3-phosphate oxidoreductase [7] and pyruvate oxidoreductase [8], and AMP-dependent (ATP-forming) kinase [9]. The occurrence of such a novel central metabolism is limited to the strains of the Thermococcales order such as T. litoralis and P. furiosus. This suggests that any specific metabolic system may be present in hyperthermophilic marine archaea. In addition, the two hyperthermophilic marine archaea have been reported to utilize peptides and pyruvate for their growth. This suggests the presence of various amino-acid and organic-acid metabolic pathways.During the course of screening for enzymes in the aminoacid and organic-acid metab...