Identification of ligand binding site of phytosulfokine receptor by on-column photoaffinity labeling

Phytosulfokine (PSK), an endogenous 5-amino-acid-secreted peptide in plants, affects cellular potential for growth via binding to PSKR1, a member of the leucine-rich repeat receptor kinase (LRR-RK) family. PSK interacts with PSKR1 in a highly specific manner with a nanomolar dissociation constant. However, it is not known which residues in the PSKR1 extracellular domain constitute the ligand binding pocket. Here, we have identified the PSK binding domain of carrot PSKR1 (DcPSKR1) by photoaffinity labeling. We cross-linked the photoactivatable PSK analog [(125)I]-[N(epsilon)-(4-azidosalicyl)Lys(5)]PSK with DcPSKR1 using UV irradiation and mapped the cross-linked region using chemical and enzymatic fragmentation. We also established a novel "on-column photoaffinity labeling" methodology that allows repeated incorporation of the photoaffinity label to increase the efficiency of the photoaffinity cross-linking reactions. We purified a labeled DcPSKR1 tryptic fragment using anti-PSK antibodies and identified a peptide fragment that corresponds to the 15-amino-acid Glu(503)-Lys(517) region of DcPSKR1 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Deletion of Glu(503)-Lys(517) completely abolishes the ligand binding activity of DcPSKR1. This region is in the island domain flanked by extracellular LRRs, indicating that this domain forms a ligand binding pocket that directly interacts with PSK.     

Chimpanzee chromosomes: retrotransposable compound repeat DNA organization (RCRO) and its influence on meiotic prophase and crossing-over

The terminal C-bands that are a specific feature of chimpanzee chromosomes were dissected using a molecular cytogenetic technique, PRINS, with primers for telomeric sequences, subterminal satellite, and retrotransposable elements (HERV-K and -W). These DNA elements jointly formed a large block of retrotransposable compound repeat DNA organization (RCRO) at the terminal C-band regions of 30 chromosomes, and are also located at the centromeric regions of some chromosomes. Additionally, a block consisting of all members of the RCRO has transposed to the middle (q31.1) of the long arm of chromosome 6, and three members, the subterminal satellite and the two HERVs, have integrated into the proximal region (q14.4) of the long arm of chromosome 14. Terminal RCROs seem to induce and prolong the bouquet stage in meiotic prophase, and to affect chiasma formation, together with interstitial RCROs. It is also postulated that RCROs may cause a position effect to gene expression, resulting in gene silencing and/or late replication.     

Biochemical characterization of the Drosophila wingless signaling pathway based on RNA interference

Regulation of Armadillo (Arm) protein levels through ubiquitin-mediated degradation plays a central role in the Wingless (Wg) signaling. Although zeste-white3 (Zw3)-mediated Arm phosphorylation has been implicated in its degradation, we have recently shown that casein kinase Ialpha (CKIalpha) also phosphorylates Arm and induces its degradation. However, it remains unclear how CKIalpha and Zw3, as well as other components of the Arm degradation complex, regulate Arm phosphorylation in response to Wg. In particular, whether Wg signaling suppresses CKIalpha- or Zw3-mediated Arm phosphorylation in vivo is unknown. To clarify these issues, we performed a series of RNA interference (RNAi)-based analyses in Drosophila S2R+ cells by using antibodies that specifically recognize Arm phosphorylated at different serine residues. These analyses revealed that Arm phosphorylation at serine-56 and at threonine-52, serine-48, and serine-44, is mediated by CKIalpha and Zw3, respectively, and that Zw3-directed Arm phosphorylation requires CKIalpha-mediated priming phosphorylation. Daxin stimulates Zw3- but not CKIalpha-mediated Arm phosphorylation. Wg suppresses Zw3- but not CKIalpha-mediated Arm phosphorylation, indicating that a vital regulatory step in Wg signaling is Zw3-mediated Arm phosphorylation. In addition, further RNAi-based analyses of the other aspects of the Wg pathway clarified that Wg-induced Dishevelled phosphorylation is due to CKIalpha and that presenilin and protein kinase A play little part in the regulation of Arm protein levels in Drosophila tissue culture cells.     

A spatiotemporal analysis of acoustic interactions between great reed warblers (Acrocephalus arundinaceus) using microphone arrays and robot audition software HARK

Acoustic interactions are important for understanding intra‐ and interspecific communication in songbird communities from the viewpoint of soundscape ecology. It has been suggested that birds may divide up sound space to increase communication efficiency in such a manner that they tend to avoid overlap with other birds when they sing. We are interested in clarifying the dynamics underlying the process as an example of complex systems based on short‐term behavioral plasticity. However, it is very problematic to manually collect spatiotemporal patterns of acoustic events in natural habitats using data derived from a standard single‐channel recording of several species singing simultaneously. Our purpose here was to investigate fine‐scale spatiotemporal acoustic interactions of the great reed warbler. We surveyed spatial and temporal patterns of several vocalizing color‐banded great reed warblers (Acrocephalus arundinaceus) using an open‐source software for robot audition HARK (Honda Research Institute Japan Audition for Robots with Kyoto University) and three new 16‐channel, stand‐alone, and water‐resistant microphone arrays, named DACHO spread out in the bird's habitat. We first show that our system estimated the location of two color‐banded individuals’ song posts with mean error distance of 5.5 ± 4.5 m from the location of observed song posts. We then evaluated the temporal localization accuracy of the songs by comparing the duration of localized songs around the song posts with those annotated by human observers, with an accuracy score of average 0.89 for one bird that stayed at one song post. We further found significant temporal overlap avoidance and an asymmetric relationship between songs of the two singing individuals, using transfer entropy. We believe that our system and analytical approach contribute to a better understanding of fine‐scale acoustic interactions in time and space in bird communities.     

Japanese families with autosomal dominant pure cerebellar ataxia map to chromosome 19p13.1-p13.2 and are strongly associated with mild CAG expansions in the spinocerebellar ataxia type 6 gene in chromosome 19p13.1.

Autosomal dominant cerebellar ataxia is a group of clinically and genetically heterogeneous disorders. We carried out genomewide linkage analysis in 15 families with autosomal dominant pure cerebellar ataxia (ADPCA). Evidence for linkage to chromosome 19p markers was found in nine families, and combined multipoint analysis refined the candidate region to a 13.3-cM interval in 19p13.1-p13.2. The remaining six families were excluded for this region. Analysis of CAG-repeat expansion in the alpha1A-voltage-dependent calcium channel (CACNL1A4) gene lying in 19p13.1, recently identified among 8 small American kindreds with ADPCA (spinocerebellar ataxia type 6 [SCA6]), revealed that 8 of the 15 families studied had similar, very small expansion in this gene: all affected individuals had larger alleles (range of CAG repeats 21-25), compared with alleles observed in neurologically normal Japanese (range 5-20 repeats). Inverse correlation between the CAG-repeat number and the age at onset was found in affected individuals with expansion. The number of CAG repeats in expanded chromosomes was completely stable within each family, which was consistent with the fact that anticipation was not statistically proved in the SCA6 families that we studied. We conclude that more than half of Japanese cases of ADPCA map to 19p13.1-p13.2 and are strongly associated with the mild CAG expansion in the SCA6/CACNL1A4 gene.     

Changes in import of non-human primates after ratification of CITES (Washington Convention) in Japan

The total yearly imports of exotic primates into Japan and the countries of origin from 1971 to 1984 were examined. The species, numbers, originating countries, and end-uses of the monkeys imported from 1981 to 1984 were also investigated. After a high plateau of imports which continued until 1980, the total numbers of monkeys imported into Japan were reduced by half from the pre-1980 level of 7,000–8,000/year to a level of 3,000–4,000/year. This drastic decrease appeared to reflect the effect of the Washington Convention ratified in 1980. The majority of species imported wereMacaca fascicularis of Southeast Asian origin, followed bySaimiri sciureus of South American origin. Indonesian and MalaysianM. fascicularis represented the major species in the decrease in monkey imports. However, imports from the Philippines have conversely increased since 1980. Imports ofS. sciureus andCallithrix from South America have remained almost unchanged. These data imply a trend in which experimental monkeys have changed from macaques to smaller New World monkeys.     

Random sequencing of cDNA libraries reveals a variety of expressed genes in cultured cells of rice (Oryza sativa L.)

Large scale sequencing of randomly selected cDNA clones was carried out to investigate the feasibility of this method for isolating plant genes. cDNA libraries were made using mRNA prepared from suspension-cultured cells of rice (Oryza sativa L.). Partial nucleotide sequences of 830 individual cDNA clones have been determined and compared with the GenBank database. Approximately 8% of the cDNA clones could be identified as particular genes. This method provides the opportunity to isolate large numbers of plant genes.     

Differentiation capabilities of human germ cell tumors

It is well known that human germ cell tumors are an excellent model to study not only differentiation capacity of tumor cells but also human normal somatic cell differentiation. A variety of polyclonal and monoclonal antibodies were developed against cell surface antigens of murine embryos and teratocarcinomas. Accumulated data has revealed that these antigens are sequentially expressed on embryonic cells in a well-programmed manner. They have also been shown to be useful markers to investigate somatic cell differentiation in fetal and adult tissue. In humans, however, little is known about the cellular differentiation mechanism in early embryos and whether they could be studied, i.e. whether they occur in human germ cell tumors. In present review, we discussed newly established monoclonal antibodies which were raised from human embryonal carcinoma cells. We have been studying differentiation capacity of human germ cell tumor cells by using these antibodies. Some of these antibodies clearly indicates their usefulness to specify the developmental stage of normal tissue.     

Human pancreatic polypeptide responsiveness to insulin-induced hypoglycemia in anorexia nervosa

Patients with anorexia nervosa occasionally suffer from hypoglycemic comas. We investigated the role of human pancreatic polypeptide (HPP) in insulin-induced hypoglycemia (0.1 U/kg of regular insulin). Ten female patients with anorexia nervosa (20.7 +/- 2.0 years, mean +/- SEM; 34.9 +/- 1.7 kg, mean +/- SEM) and 8 age-matched female controls (20.9 +/- 0.6 years, 51.5 +/- 0.8 kg) were tested. In the patients with anorexia nervosa, testing was performed before and after the restoration of body weight (45.0 +/- 0.8 kg). There was no significant difference in glucose nadir between patients with anorexia nervosa and the control subjects. However, glucose recovery from nadir was delayed in patients with anorexia nervosa. In anorexia nervosa patients, the plasma pancreatic glucagon responses to insulin-induced hypoglycemia did not differ from those of the controls. Results also showed, however, that HPP responses to insulin-induced hypoglycemia were significantly higher in patients with anorexia nervosa than in controls (p less than 0.01). The increased HPP responses were still present after the restoration of body weight in anorexia nervosa patients. A complete body weight recovery or a longer period of time may be required to normalize the HPP response to insulin-induced hypoglycemia in patients with anorexia nervosa, after the restoration of body weight.