Differential expression of two glucuronyltransferases synthesizing HNK-1 carbohydrate epitope in the sublineages of the rat myogenic progenitors

HNK-1 epitope is a cell-surface carbohydrate mediating various cell-cell or cell-substrate interactions. We found HNK-1 epitope in longitudinally arrayed fibers in the subpopulation of the epaxial myotome, and hypaxial myoblasts migrating into the limb bud in the rat embryo. We next investigated the expression patterns of genes encoding two glucuronyltransferases (GlcAT-P, GlcAT-D) and sulfotransferase (Sul-T), which are required for biosynthesis of HNK-1 epitope. GlcAT-P gene was expressed in the non-migrating longitudinal fibers, whereas GlcAT-D gene was expressed in the migrating myoblasts in the limb bud. Sul-T gene expression was ubiquitously observed in all these myogenic populations. Thus, differential expression of GlcAT genes may relate to the epaxial/hypaxial or migrating/non-migrating myoblast lineages.     

Immunohistochemical demonstration of Angiopoietin-2 in lymphatic vascular development

The cellular expression of Angiopoietin-2 (Ang2) was studied during lymphatic development in mouse by immunohistochemistry and compared to that of lymphatic endothelial markers. At the earliest stage of lymphvasculogenesis, Prox1-identified lymphatic precursor cells of the cardinal vein displayed an intense immunoreaction for Ang2 in their cytoplasm, implying that Ang2 may adjust lymphatic specification and sprouting from the veins under the control of Prox1. Thereafter, Ang2 was constantly expressed in Prox1 and/or LYVE-1-immunopositive endothelial cells of lymphatic sacs and vessels, ranging from lymphatic capillaries to collectors, throughout embryonic and neonatal development, and the lymphatic endothelial cells simultaneously exhibited immunoreactivity to Tie2, a primary receptor for angiopoietins. These results suggest that lymphatic endothelial cells may regulate lymphatic development via their own Ang2-Tie2 signaling. Ang2 is further immunolocalized in the developing blood vessels including hepatic sinusoids, adrenal medullary vasculature and postnatal pulmonary vessels, thereby indicating that the blood vessels, which undergo vascular remodeling and sudden alteration of blood flow during the development, are also likely to express Ang2. The present study is first to demonstrate Ang2 expression in the lymphatic endothelial cells during development, and consequently Ang2 is regarded as a molecular profile of the developing lymphatic endothelial cells required for lymphatic vascular organization.     

A dominant function of CCaMK in intracellular accommodation of bacterial and fungal endosymbionts

In legumes, Ca²⁺/calmodulin‐dependent protein kinase (CCaMK) is a component of the common symbiosis genes that are required for both root nodule (RN) and arbuscular mycorrhiza (AM) symbioses and is thought to be a decoder of Ca²⁺ spiking, one of the earliest cellular responses to microbial signals. A gain‐of‐function mutation of CCaMK has been shown to induce spontaneous nodulation without rhizobia, but the significance of CCaMK activation in bacterial and/or fungal infection processes is not fully understood. Here we show that a gain‐of‐function CCaMK^T265D suppresses loss‐of‐function mutations of common symbiosis genes required for the generation of Ca²⁺ spiking, not only for nodule organogenesis but also for successful infection of rhizobia and AM fungi, demonstrating that the common symbiosis genes upstream of Ca²⁺ spiking are required solely to activate CCaMK. In RN symbiosis, however, CCaMK^T265D induced nodule organogenesis, but not rhizobial infection, on Nod factor receptor (NFRs) mutants. We propose a model of symbiotic signaling in host legume plants, in which CCaMK plays a key role in the coordinated induction of infection thread formation and nodule organogenesis.     

A polymerase chain reaction-based ribosomal DNA detection technique using a surface plasmon resonance detector for a red tide causing microalga, Alexandrium affine

A detection technique with a DNA probe was developed for the bloom‐forming alga Alexandrium affine harvested in Japan. The design of this probe was based on the sequence polymorphism within the 28S ribosomal DNA (rDNA) of this strain using the BIAcore? 2000 biosensor, which determines surface plasmon resonance. The specific DNA sequence in 28S rDNA for A. affine was determined by sequence data analysis, and a probe was designed for the detection of A. affine. A fragment of the 28S rDNA from A. affine was amplified by polymerase chain reaction and applied to the BIAcore? sensor system, and the target DNA was selectively recognized by species‐specific hybridization using two DNA probes: a fluorescein isothiocyanate (FITC)‐labeled probe and a biotin‐labeled DNA probe. Using FITC‐labeled anti‐immunogloblin G antibody, enhancement of the response for the target DNA can be detected directly as a resonant unit change. In this detection method, a difference within only 20 base pairs of the target could be detected, and specific detection of A. affine was achieved intraspecifically.     

Radiosynthesis and evaluation of new PET ligands for peripheral cannabinoid receptor type 1 imaging

Cannabinoid receptor type 1 (CB1) is mainly expressed in the brain, as well as being expressed in functional relevant concentrations in various peripheral tissues. 1-(4-Chlorophenyl)-3-(3-(6-(pyrrolidin-1-yl)pyridin-2-yl)phenyl)urea (PSNCBAM-1, 1) was developed as a potent allosteric antagonist for CB1 and its oral administration led to reductions in the appetite and body weight of rats. Several analogs of 1 (compounds 2 and 3) were recently identified through a series of structure-activity relationship studies. Herein, we report the synthesis of radiolabeled analogs of these compounds using [¹¹C]COCl₂ and an evaluation of their potential as PET ligands for CB1 imaging using in vitro and in vivo techniques. [¹¹C]2 and [¹¹C]3 were successfully synthesized in two steps using [¹¹C]COCl₂. The radiochemical yields of [¹¹C]2 and [¹¹C]3 were 17 ± 8% and 20 ± 9% (decay-corrected to the end of bombardment, based on [¹¹C]CO₂). The specific activities of [¹¹C]2 and [¹¹C]3 were 42 ± 36 and 37 ± 13 GBq/μmol, respectively. The results of an in vitro binding assay using brown adipose tissue (BAT) homogenate showed that the binding affinity of 2 for CB1 (K_D = 15.3 µM) was much higher than that of 3 (K_D = 26.0 µM). PET studies with [¹¹C]2 showed a high uptake of radioactivity in BAT, which decreased in animals pretreated with AM281 (a selective antagonist for CB1). In conclusion, [¹¹C]2 may be a useful PET ligand for imaging peripheral CB1 in BAT.     

The ORC1 cycle in human cells: I. cell cycle-regulated oscillation of human ORC1

Components of ORC (the origin recognition complex) are highly conserved among eukaryotes and are thought to play an essential role in the initiation of DNA replication. The level of the largest subunit of human ORC (ORC1) during the cell cycle was studied in several human cell lines with a specific antibody. In all cell lines, ORC1 levels oscillate: ORC1 starts to accumulate in mid-G1 phase, reaches a peak at the G1/S boundary, and decreases to a basal level in S phase. In contrast, the levels of other ORC subunits (ORCs 2-5) remain constant throughout the cell cycle. The oscillation of ORC1, or the ORC1 cycle, also occurs in cells expressing ORC1 ectopically from a constitutive promoter. Furthermore, the 26 S proteasome inhibitor MG132 blocks the decrease in ORC1, suggesting that the ORC1 cycle is mainly due to 26 S proteasome-dependent degradation. Arrest of the cell cycle in early S phase by hydroxyurea, aphidicolin, or thymidine treatment is associated with basal levels of ORC1, indicating that ORC1 proteolysis starts in early S phase and is independent of S phase progression. These observations indicate that the ORC1 cycle in human cells is highly linked with cell cycle progression, allowing the initiation of replication to be coordinated with the cell cycle and preventing origins from refiring.     

Conditioned olfactory responses of a predatory mite, Neoseiulus womersleyi, to volatiles from prey-infested plants

To clarify the prey‐finding behavior of the predatory mite Neoseiulus womersleyi (Schicha) (Acari: Phytoseiidae), we studied its olfactory responses to volatiles from the prey‐infested plant on which the mites had been collected. We used a local N. womersleyi population called Kanaya collected from tea (Camellia sinensis L.) (Theaceae) plants infested by Tetranychus kanzawai Kishida (Acari: Tetranychidae) in Kanaya City, Japan. Neoseiulus womersleyi (Kanaya population) were more attracted to volatiles from tea plants infested with five female T. kanzawai per leaf for 7 days than to intact tea leaves in a Y‐tube olfactometer. Tetranychus kanzawai‐induced tea leaf volatiles were identified as (E)‐β‐ocimene, (E)‐4,8‐dimethyl‐1,3,7‐nonatriene, and (E,E)‐α‐farnesene. As olfactory responses are known to differ among local populations of N. womersleyi, we compared the responses of the Kanaya population with those of a Kikugawa population collected from tea plants infested by T. kanzawai in Kikugawa City. To test the influence of previous predation experience, we reared the two populations on tea plants infested by T. kanzawai or on kidney bean plants (Phaseolus vulgaris) infested by Tetranychus urticae Koch. The Kanaya population was more attracted to the volatiles from infested plants on which they had been reared. Because the Kanaya population was not attracted to the plant volatiles they had not previously experienced, the positive response to previously experienced volatiles might be the result of learning. By contrast, the Kikugawa population showed no preference for previously experienced volatiles from infested plants. The implications of this flexibility in foraging behavior are discussed.     

Time allocation of Orius sauteri in attacking Thrips palmi on an eggplant leaf

Orius sauteri (Poppius) (Heteroptera: Anthocoridae) is a polyphagous predator used as a biological control agent of palm thrips, Thrips palmi (Karny) (Thysanoptera: Thripidae). We studied O. sauteri's searching efficiency, time allocation on a leaf, leaving tendency, and attacking of prey. Approximately 78% of the encountered prey was eaten. Searching for prey was concentrated for 86% of the time on the lower leaf side, where palm thrips are usually found. Patch residence times on empty leaves were different from those on leaves with T. palmi larvae. Walking activity was not affected by the thrips density, and walking took place during 64% of the total search time. The leaving tendency of O. sauteri was affected by the time from patch entry and the presence or absence of palm thrips, but not by the thrips density. If prey were present, the leaving tendency decreased (multiplication factor 0.327), resulting in longer giving‐up times than when no prey was present. The fact that the leaving tendency increases when patch exploitation lasts longer suggests that not much time is wasted on patches where encounters with prey are scarce.