Development of a cell-based assay for ecdysteroid quantification using an early ecdysteroid-inducible gene promoter

Ecdysteroids, primarily 20-hydroxyecdysone (20E) and ecdysone (E), are steroid hormones that regulate various developmental and physiological processes in insects. Commonly, immunoassays are used to quantify ecdysteroid titers of insects. However, the antibodies used in these assays react not only with 20E and E but often also with their inactive reserves and metabolites, and thus require purification before they can be quantified precisely. Here, we developed a simple cell-based method to quantify only the hormonally active ecdysteroids using newly established cells harboring the firefly luciferase gene under the control of the ecdysteroid-inducible promoter of the E75A gene of the silkworm Bombyx mori L. These cells also constitutively expressed the Renilla luciferase gene using the baculovirus ie2 promoter for internal reference. This cell-based method detected hormonally active ecdysteroids with significantly higher sensitivity than their inactive metabolites. Hemolymph ecdysteroid titers, determined using a dual luciferase assay after exposing these cells to crude extracts of B. mori larval and pupal hemolymph, agreed well with the sum of the 20E and E titers, which were quantified individually using a radioimmunoassay after they had been separated by HPLC. Thus, this method is very useful for quantifying the ecdysteroid titers of insects, particularly when the samples contain large amounts of ecdysteroid reserves and metabolites.     

Chimpanzee mothers at Bossou, Guinea carry the mummified remains of their dead infants

The forests surrounding Bossou, Guinea, are home to a small, semi-isolated chimpanzee community studied for over three decades [1]. In 1992, Matsuzawa [2] reported the death of a 2.5-year-old chimpanzee (Jokro) at Bossou from a respiratory illness. The infant's mother (Jire) carried the corpse, mummified in the weeks following death, for at least 27 days. She exhibited extensive care of the body, grooming it regularly, sharing her day- and night-nests with it, and showing distress whenever they became separated. The carrying of infants' corpses has been reported from a number of primate species, both in captivity and the wild [3-7] - albeit usually lasting a few days only - suggesting a phylogenetic continuity for a behavior that is poignant testament to the close mother-infant bond which extends across different primate taxa. In this report we recount two further infant deaths at Bossou, observed over a decade after the original episode but with striking similarities.     

Jumping Transmission of Action Potential between Separately Placed Internodal Cells of Chora corallina

We report here a new type of cell-to-cell communication. Wenoticed that a characean cell can transmit its action potentialsto one or more cells which lie parallel to it separately inartificial pond water without any special connection betweenthe cells. Two experimental facts proved this transmission notchemical but electrical. No transmission was observed firstwhen an Ag-AgCl wire was placed between two cells, and secondwhen the whole length of the first cell was stimulated so thatnon-propagated action potential was induced. The results showthat the traveling electrical field strength might be the fundamentalfactor in the jumping transmission. (Received November 10, 1989; Accepted December 22, 1989)     

Elevated levels of oxidative DNA damage activate p53 and caspases in brain of ayu with aging

It is well known that ayu (Plecoglossus altivelis) die after spawning. Their lifespan is known to be only 1 year; possibly one contributing factor to post‐spawning mortality in ayu is the enhanced oxidative stress, probably inducing DNA damage and subsequent DNA repair systems (i.e. phosphorylated p53), which in turn may cause apoptosis and a shortened lifespan. To examine this possibility, we surveyed p53 and its phosphorylation state, oxidative DNA damage by measuring the levels of 8‐hydroxy‐2′‐deoxyguanosine, and the induction of apoptosis by measuring levels of caspase‐3, ‐9/6 in the brain at different stages. Accumulation of oxidative stress in brain DNA was accompanied by caspase‐3, ‐9/6, and stimulates p53 through the phosphorylation of this p53 (specifically residue Ser 15) in ayu brain with aging.     

Virological and immunological bases for HIV-1 vaccine design

A logical approach for prophylactic HIV-1 vaccine development begins by recognition that the regimen needs to contain viruses which are not cleared by primary host immune responses and develop persistent infection. Hence the required strategy is different from the one against self-remitting acute infections which aims at eliciting robust host immune responses in advance by infection mimicry. Host adaptive immune responses do play a central role in primary resolution from acute HIV-1 and simian immunodeficiency virus (SIV) infection, but as observed in the non-remitting disease course, their function is not fully exerted, leading to failure in viral containment. Either overcoming the limitations of antiviral immunity in natural infection or augmenting the effectors potentially capable of controlling virus replication would be essential for development of an effective HIV-1 vaccine. This approach is hand-in-hand with understanding of the reversibility of various steps leading to establishment of persistent HIV-1 infection. This article reviews the interplay between HIV-1/SIV and the infected host, mainly focusing on macaque models of SIV infection and characterization of the two major wings of adaptive immunity, cytotoxic T lymphocytes (CTLs) and neutralizing antibodies. Discussed in parallel are the up-to-date topics of HIV-1 vaccine development including our recent progress.     

Defect of human immunodeficiency virus type 2 Gag assembly in Saccharomyces cerevisiae

We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly.     

Phenosite: a web database integrating the mouse phenotyping platform and the experimental procedures in mice

Recently, a number of collaborative large-scale mouse mutagenesis programs have been launched. These programs aim for a better understanding of the roles of all individual coding genes and the biological systems in which these genes participate. In international efforts to share phenotypic data among facilities/institutes, it is desirable to integrate information obtained from different phenotypic platforms reliably. Since the definitions of specific phenotypes often depend on a tacit understanding of concepts that tends to vary among different facilities, it is necessary to define phenotypes based on the explicit evidence of assay results. We have developed a website termed PhenoSITE (Phenome Semantics Information with Terminology of Experiments: http://www.gsc.riken.jp/Mouse/), in which we are trying to integrate phenotype-related information using an experimental-evidence-based approach. The site's features include (1) a baseline database for our phenotyping platform; (2) an ontology associating international phenotypic definitions with experimental terminologies used in our phenotyping platform; (3) a database for standardized operation procedures of the phenotyping platform; and (4) a database for mouse mutants using data produced from the large-scale mutagenesis program at RIKEN GSC. We have developed two types of integrated viewers to enhance the accessibility to mutant resource information. One viewer depicts a matrix view of the ontology-based classification and chromosomal location of each gene; the other depicts ontology-mediated integration of experimental protocols, baseline data, and mutant information. These approaches rely entirely upon experiment-based evidence, ensuring the reliability of the integrated data from different phenotyping platforms.     

Ground‐nesting by the chimpanzees of the Nimba Mountains,Guinea: environmentally or socially determined?

The chimpanzees (Pan troglodytes verus) of the Nimba Mountains, Guinea, West Africa, commonly make both elaborate ("night") and simple ("day") nests on the ground. In this study we investigated which factors might influence ground-nesting in this population, and tested two ecological hypotheses: 1) climatic conditions, such as high wind speeds at high altitudes, may deter chimpanzees from nesting in trees; and 2) a lack of appropriate arboreal nesting opportunities may drive the chimpanzees to nest on the ground. In addition to testing these two hypotheses, we explored whether ground-nesting is a sex-linked behavior. Data were collected monthly between August 2003 and May 2004 along transects and ad libitum. To identify the sex of ground-nesting individuals, we used DNA extracted from hair samples. The results showed that the occurrence and distribution of ground nests were not affected by climatic conditions or a lack of appropriate nest trees. Support was found for the notion that ground-nesting is a sex-linked behavior, as males were responsible for building all of the elaborate ground nests and most of the simple ground nests sampled. Elaborate ground nests occurred mostly in nest groups associated with tree nests, whereas simple ground nests usually occurred without tree nests in their vicinity. These results suggest that ground-nesting may be socially, rather than ecologically, determined.