New method for forming large embryoid bodies using the wall of the culture dish along with an analysis of their structural characteristics

Early embryonic stem (EES) cells, which were established from 2 cell stage embryos obtained from ddY mice, had similar characteristics as embryonic stem (ES) cells. These cells were maintained in an undifferentiated stage in growth media supplemented with leukemia inhibitory factor (LIF) and were capable of differentiating into triploblastic tissues under various growth factors. It has been known that normal sized embryoid bodies (EBs) are formed by removing LIF. In this study, large EBs gradually formed along the side wall of a culture dish, particularly at the boundary between the air and the growth medium when cells were cultured for a considerable period of time and without subculturing. We call this method the “wall adhesion culture” procedure. The method itself is simple and do not need any instruments except plastic dishes because only the side walls of the dishes were utilized. The mean thickness of the large EBs was about 1.5 mm 3 months after establishing the static culture. Their surface was covered with a monolayer of cells and they contained an eosinophilic cell matrix. By electron microscopy, some characteristic structures was observed, such as intracisternal A particles which were present inside the swelling of the rough endoplasmic reticulum. Since many tissues derived from ES cells are obtained through EBs, it is expected that efficient acquisition of sufficient quantities of these structures using the wall adhesion culture procedure will be a shortcut for using ES cells in regenerative medicine.     

Up-regulation of genes related to the ubiquitin-proteasome system in the brown adipose tissue of 24-h-fasted rats

The functional balance between brown adipose tissue (BAT) and white adipose tissue (WAT) is important for metabolic homeostasis. We compared the effects of fasting on the gene expression profiles in BAT, WAT and liver by using a DNA microarray analysis. Tissues were obtained from rats that had been fed or fasted for 24 h. Taking the false discovery rate into account, we extracted the top 1,000 genes that had been differentially expressed between the fed and fasted rats. In all three tissues, a Gene Ontology analysis revealed that the lipid and protein biosynthesis-related genes had been markedly down-regulated. The whole-body fuel shift from glucose to triacylglycerol and the induction of autophagy were also observed. There was marked up-regulation of genes in the 'protein ubiquitination' category particularly in BAT of the fasted rats, suggesting that the ubiquitin-proteasome system was involved in saving energy as an adaptation to food shortage.     

Identification of new amine acceptor protein substrate candidates of transglutaminase in rat liver extract: use of 5-(biotinamido) pentylamine as a probe

Transglutaminases (TGs) are a family of enzymes that catalyze Ca(2+)-dependent post-translational modification of proteins by introducing protein-protein crosslinks (between specific glutamine and lysine residues), amine incorporation, and site-specific deamidation. In this study, new amine acceptor protein substrates of TG were isolated from rat liver extract and identified using 5-(biotinamido) pentylamine, a biotinylated primary amine substrate, as a probe. TG protein substrate candidates labeled with biotin by endogenous TG activity were isolated and recovered by avidin column chromatography. Proteins with molecular masses of 40, 42, and 45 kDa were the main components of the labeled proteins. Determination of their partial amino acid sequences and immunoblotting analyses were done to identify them. The 45-kDa protein was identical with betaine-homocysteine S-methyltransferase (EC 2.2.2.5), which was identified in our previous study. The 40- and 42-kDa proteins were identified as arginase-I (EC 3.5.3.1) and fructose-1,6-bisphosphatase (EC 3.1.3.11) respectively. TG catalyzed incorporation of 5-(biotinamido) pentylamine into both arginase-I and fructose-1,6-bisphosphatase purified from rat liver was confirmed in vitro. These results suggest that these two enzymes are the new protein substrate candidates of TG and that they can be modified post-translationally by cellular TG.     

Arginine increases the solubility of coumarin: comparison with salting-in and salting-out additives

Poor aqueous solubility of low molecular weight drug substances hampers their development as pharmacological agents. Here, we have examined the effects of arginine on the solubility of organic compounds, coumarin, caffeine and benzyl alcohol, in aqueous solution. Arginine increased the solubility of aromatic coumarin, but not non-aromatic caffeine, concentration dependently, suggesting the favourable interaction of arginine with the aromatic structure. Consistent with this, arginine also increased the solubility of aromatic benzyl alcohol. Guanidine hydrochloride, urea and salting-in salts increased both coumarin and caffeine solubilities, while salting-out salts decreased them. These results suggest the specific interaction of arginine with aromatic groups, leading to increased solubility of coumarin. However, the effect of 1 M arginine on coumarin solubility was at most approximately 2-fold, which may limit its applications as a solubility enhancing agent.     

The assembly of the FtsZ ring at the mid-chloroplast division site depends on a balance between the activities of AtMinE1 and ARC11/AtMinD1

Chloroplast division comprises a sequence of events that facilitatesymmetric binary fission and that involve prokaryotic-like stromaldivision factors such as tubulin-like GTPase FtsZ and the divisionsite regulator MinD. In Arabidopsis, a nuclear-encoded prokaryoticMinE homolog, AtMinE1, has been characterized in terms of itseffects on a dividing or terminal chloroplast state in a limitedseries of leaf tissues. However, the relationship between AtMinE1expression and chloroplast phenotype remains to be fully elucidated.Here, we demonstrate that a T-DNA insertion mutation in AtMinE1results in a severe inhibition of chloroplast division, producingmotile dots and short filaments of FtsZ. In AtMinE1 sense (overexpressor)plants, dividing chloroplasts possess either single or multipleFtsZ rings located at random intervals and showing constrictiondepth, mainly along the chloroplast polarity axis. The AtMinE1sense plants displayed equivalent chloroplast phenotypes toarc11, a loss-of-function mutant of AtMinD1 which forms replicatingmini-chloroplasts. Furthermore, a certain population of FtsZrings formed within developing chloroplasts failed to initiateor progress the membrane constriction of chloroplasts and consequentiallyto complete chloroplast fission in both AtMinE1 sense and arc11/atminD1plants. Our present data thus demonstrate that the chloroplastdivision site placement involves a balance between the opposingactivities of AtMinE1 and AtMinD1, which acts to prevent FtsZring formation anywhere outside of the mid-chloroplast. In addition,the imbalance caused by an AtMinE1 dominance causes multiple,non-synchronous division events at the single chloroplast level,as well as division arrest, which becomes apparent as the chloroplastsmature, in spite of the presence of FtsZ rings.     

Induction of serotonin accumulation by feeding of rice striped stem borer in rice leaves

Tryptophan (Trp)-related secondary metabolism has been implicated in the defense against pathogen infection and insect feeding in various gramineous species. Recently, we also reported that rice plant accumulated serotonin and tryptamine as well as their amide compounds coupled with phenolic acids in response to the infection by fungal pathogen. These compounds were likely to play an important role in the formation of physical barrier to the invading pathogens. To extend our study to elucidate the defensive role of Trp-derived secondary metabolism in gramineous plants, we examined in this study whether it is activated in response to herbivore attack as well. Third leaves of rice plant were fed on by third instar larvae of rice striped stem borer for 24 h or 48 h. The analysis of four Trp-derived metabolites including tryptamine, serotonin feruloyltryptamine (FerTry) and p-coumaroylserotonin (CouSer) by liquid chromatography coupled with tandem mass spectrometry revealed that their contents clearly increased in response to the larvae feeding. The respective amounts of tryptamine, serotonin, FerTry and CouSer in the larvae-fed leaves were 12-, 3.5-, 33- and 140-fold larger than those in control leaves 48 h after the start of feeding.Key words: rice, Oryza sativa, Gramineae, serotonin, secondary metabolism, rice striped stem borer, Chilo suppressalisPlants defend themselves from environmental stresses by utilizing secondary metabolism. One of major biological stresses that plants have to cope with is attack by herbivorous insects. In the interactions with herbivorous insects, various secondary metabolites that are derived from tryptophan (Trp) pathway have been shown to play defensive roles in plants including gramineous species. For example, benzoxazinone glucosides in wheat (Triticum aestivum), rye (Secale sereale) and maize (Zea mays) express toxic and antifeeding effects on herbivorous insects.^1,2 Benzoxazinones are biosynthesized from indole-3-glycerol phosphate, an intermediate of Trp synthesis.^3,4 Another example of those compounds is gramine in barley (Hordeum vulgare). Gramine is a Trp-rerived indole amine,⁵ and has been received attention in the resistance mainly against aphids on the basis of its toxicity and deterrence.⁶We recently found that Trp-derived secondary metabolism is also involved in defense responses of rice (Oryza sativa) leaves to infection by brown spot fungus (Bipolaris oryzae).⁷ The infection of the fungus activates Trp biosynthesis and accumulation of serotonin and of smaller amounts of tryptamine, feruloyltryptamine (FerTry) and p-coumaroylserotonin (CouSer). In addition, the enhancement of serotonin peroxidase activity and incorporation of serotonin in the cell walls were detected. Thus, it is very likely that that serotonin-derived materials deposit in cell walls after oxidative polymerization to constitute a part of physical defense system of rice, which may be reminiscent of the wound sloughing in animals. These findings prompted us to investigate whether Trp-related secondary metabolism is also involved in the defense of rice plant against the attack by insects, as in the cases of other gramineous plants mentioned above. While the response of plants to pathogenic infection is generally different from that to insect herbivory, Trp-derived secondary metabolites have occasionally been implicated in both responses.^8–10 Here, we report the results of our study to examine the effects of herbivory by rice striped stem borer (Chilo suppressalis) on the Trp derived secondary metabolism in rice leaves.Rice (cv. Nipponbare) leaves were incubated with larvae of C. suppressalis in a feeding tube assembled according to Oikawa et al.,⁸ Aerial parts of two 12-day-old rice seedlings were excised, and their cutting ends were immersed in distilled water in a vial. Three third instar larvae of C. suppressalis were put on the leaves, and the leaves with larvae were covered by a plastic tube. For comparison, the control leaves were wounded by razor blade at the start of the incubation. After incubation for 24 h or 48 h with 16/8 h LD cycle at 28°C, the leaves were extracted with 10 volumes of 80% methanol, and analyzed by liquid chromatography coupled with tandem mass spectrometry in multiple reaction monitoring mode.As shown in Figure 1, the contents of tryptamine and serotonin increased along with time in the larvae-fed leaves. The respective contents of tryptamine and serotonin in the leaves were 12- and 3.5-fold larger than those in control leaves 48 h after the start of feeding. The accumulation of FerTry and CouSer was also observed after larvae feeding with the contents being 33- and 140-fold larger than those in control leaves, respectively. Their contents, however, were approximately 10-fold smaller than the corresponding amines.Open in a separate windowFigure 1Accumulation of Trp derived metabolites in the leaves attacked by rice striped stem borer. Chemical structures of analyzed compounds (A). The contents of tryptamine (B), serotonin (B), FerTry (C) and CouSer (D) were determined by LC-MS/MS analysis. The third leaves of 12-d-old rice seedlings were fed on by rice striped stem borer (brack bars) or wounded by razor blade as control (white bars). After incubation, the leaves were extracted by 80% methanol. The contents of metabolites at time 0 are represented as gray bars.In the interaction of rice plant with B. oryzae, serotonin was shown to be incorporated into cell walls as a part of physical defense system.⁷ In an analogous way, modification of cell walls by serotonin might function in sealing the sites injured by insect feeding to protect the leaves from desiccation, and opportunistic and insect-mediated infection by microorganisms. Indeed, at the cutting edge of the leaves, the formation of brown materials was observed. In addition, since serotonin is a neurotransmitter of insects and tryptamine has been indicated to be a neuroactive substance, their accumulation might directly affect behavior and physiology of some insects. High concentrations of tryptamine have been shown to express anti-oviposition activity toward Bemisia tabaci¹¹ and anti-feeding activities toward Malacosoma disstria and Manduca sexta.¹²The low levels of serotonin, tryptamine and their amides in the control leaves suggest that these compounds are induced in response to some components produced during the interaction between the plant and the herbivore. In this relation, it has been shown that elicitors are present in the saliva of some herbivous insects, which induce volatile emission from the plant to attracts their natural enemies.^13,14 Induction of Trp-derived secondary metabolites by the herbivore attack may likely be a result of recognition of some insect-derived molecules by rice leaves, similarly to the induction of volatile emission.The induced accumulation of indole amines and their hydroxycinnamic acid amides in the rice leaves attacked by C. suppressalis suggests that a common signaling pathway might be involved in the responses to pathogen infection and insect feeding. However, the composition of induced compounds was different between the responses to the two biological stresses. The content of tryptamine in the larvae-fed leaves was comparable to that reported in the B. oryzae-infected leaves, whereas the amount of serotonin (approximately 35 nmol/gFW) was much smaller than that in the infected leaves (approximately 250 nmol/gFW). This characteristic was similar to the response of rice leaves to methyl jasmonate (MeJA), which also induced accumulation of these Trp-derived secondary metabolites.⁷ The strong activation of the conversion of tryptamine to serotonin may require infection-specific signals.The serotonin accumulation in rice appears to be similar to the accumulation of gramine in barley in several aspects. Gramine accumulation has been demonstrated to be induced by either infection by pathogens⁹ or infestation by the aphid Schizaphis graminum.¹⁰ In addition, the gene encoding N-methyltransferase that catalyzes the final reaction in the gramine biosynthetic pathway is upregulated by MeJA, suggesting gramine synthesis is at least partly under the control of jasmonate signaling pathway.^15,16 The inducible serotonin production may be an archetypal form of the biosynthesis of more complicated indole amine in barley.     

Comprehensive analysis of glycosyltransferases in eukaryotic genomes for structural and functional characterization of glycans

Glycosyltransferases comprise highly divergent groups of enzymes, which play a central role in the synthesis of complex glycans. Because the repertoire of glycosyltransferases in the genome determines the range of synthesizable glycans, and because the increasing amount of genome sequence data is now available, it is essential to examine these enzymes across organisms to explore possible structures and functions of the glycoconjugates. In this study, we systematically investigated 36 eukaryotic genomes and obtained 3426 glycosyltransferase homologs for biosynthesis of major glycans, classified into 53 families based on sequence similarity. The families were further grouped into six functional categories based on the biosynthetic pathways, which revealed characteristic patterns among organism groups in the degree of conservation and in the number of paralogs. The results also revealed a strong correlation between the number of glycosyltransferases and the number of coding genes in each genome. We then predicted the ability to synthesize major glycan structures including N-glycan precursors and GPI-anchors in each organism from the combination of the glycosyltransferase families. This indicates that not only parasitic protists but also some algae are likely to synthesize smaller structures than the structures known to be conserved among a wide range of eukaryotes. Finally we discuss the functions of two large families, sialyltransferases and β4-glycosyltransferases, by performing finer classifications into subfamilies. Our findings suggest that universality and diversity of glycans originate from two types of evolution of glycosyltransferase families, namely conserved families with few paralogs and diverged families with many paralogs.     

Therapeutic Strategy for Ischemic Stroke

Possible strategies for treating ischemic stroke include: (1) Neuroprotection: preventing damaged neurons from undergoing apoptosis in the acute phase of cerebral ischemia; (2) Stem cell therapy: the repair of broken neuronal networks with newly born neurons in the chronic phase of cerebral ischemia. Firstly, we studied the neuroprotective effect of a calcium channel blocker, azelnidipine, or a by-product of heme degradation, biliverdin, in the ischemic brain. These results revealed both azelnidipine and biliverdin had a neuroprotective effect in the ischemic brain through their anti-oxidative property. Secondly, we investigated the role of granulocyte colony-stimulating factor (G-CSF) by administering G-CSF to rats after cerebral ischemia and found G-CSF plays a critical role in neuroprotection. Lastly, we developed a restorative stroke therapy with a bio-affinitive scaffold, which is able to provide an appropriate environment for newly born neurons. In the future, we will combine these strategies to develop more effective therapies for treatment of strokes. Special issue article in honor of Dr. Akitane Mori.