Variability in Toxicity of the Dinoflagellate Alexandrium Tamarense Isolated from Hiroshima Bay, Western Japan, as a Reflection of Changing Environmental Conditions

The variability of cellular toxin content in the dinoflagellateAlexandrium tamarense isolated from Hiroshima Bay was analyzedunder a variety of culture conditions. Growth and toxicity wererepresented as a function of light (80, 90, 110, 160 and 350µmol m^–2 s ^–1), temperature (12, 17 and 22°C),salinity (13, 16.5, 19.5, 25, 29, 33, 36.5 and 38 PSU) and ammoniumconcentration (0.11, 0.22 and 0.44 mM). Toxicity was measuredby the tissue culture bioassay using mouse neuroblastoma cells,and expressed as saxitoxin concentration equivalents. Cellulartoxicity increased with decreasing salinity. At temperaturesof 17 and 22°C, maximum toxin content was observed at thelowest light intensity and growth rate. At the lowest temperatureof 12°C, maximum toxin content was observed at intermediatelight intensities and growth rates. A drastic increase in toxincontent with an increase in ammonium concentration from 0.11to 0.22 mM supported the idea that ammonium utilization fortoxin production directly brings about a high toxin contentinA. tamarense. Our results ecologically imply that the cellsbecome highly toxic in environments with low salinity and highammonium concentration, and successive cloudy days. Such environmentalconditions may lead to increasing risk of shellfish toxification.     

Transient gene suppression in a red alga, Cyanidioschyzon merolae 10D

Antisense suppression is a powerful tool to analyze gene function. In this study, we show that antisense RNA suppressed the expression of a target gene in the unicellular red alga, Cyanidioschyzon merolae. In this study, the antisense strand of the catalase gene was cloned and inserted into an expression vector upstream of the GFP gene. This plasmid was introduced into C. merolae cells using a polyethylene glycol-mediated transformation protocol. Using the expression of GFP as a marker of transformed cells, the expression of catalase was examined by immunocytochemistry. Decreased expression of catalase was observed in cells that were transformed with the antisense strand of the catalase gene. These results indicate the utility of this antisense suppression system.     

Cloning and sequence determination of the gene encoding the largest subunit of the fission yeast Schizosaccharomyces pombe RNA polymerase I

The gene encoding the largest subunit of RNA polymerase I (SPRPA190) was cloned from the fission yeast Schizosaccharomyces pombe by cross-hybridization with a probe containing part of the corresponding Saccharomyces cerevisiae gene RPA190. The SPRPA190 gene is present in a single copy per haploid genome and is essential for cell growth. The polypeptide encoded by this gene, as deduced from the nucleotide sequence of the uninterrupted coding frame, consists of 1689 amino acids and its calculated Mr is 189,300. The amino acid identity between the subunits of the two yeast species is 50%. Amino acid sequence conservation covers the regions previously suggested to be functionally important for the S. cerevisiae enzyme. In addition, two markedly hydrophilic regions recognized in the S. cerevisiae polypeptide can also be recognized in the S. pombe polypeptide in approximately the same positions, even though the amino acid sequences in these regions are diverged from each other. In the 5'-flanking region of the gene, several nucleotide sequence elements are detected which are also found in the two S. pombe ribosomal protein genes so far sequenced.     

Meteorology and species composition of plant communities, birds and fishes before and after initial impoundment of Miharu Dam Reservoir, Japan

The immediate impact of damming appears most notably at the first filling of water, when the dam blocks the river and a lake suddenly forms. In this review, the changes in meteorology, plant communities, birds and fishes surrounding initial impoundment of Miharu Dam, constructed in an Asian Monsoon region, are summarised based on previous papers and subsequent field research. Although wind and temperature changes were investigated, land and lake wind occur due to the different thermal properties between the land and lake, and this type of wind often occurs at large lakes such as Glen Canyon Dam Reservoir or Lake Biwa. The size of Miharu Dam Reservoir (ponding area 2.9 km²) was insufficient to cause land–lake air differentials. Therefore, wind direction and air temperature were unaffected. Mountain winds weakened at the lake centre and near the dam body. Changes in vegetation were especially diverse at the drawdown zone (the slopes above and below the normal water level). On slopes above this zone, trees died and species composition changed due to submergence. Within the drawdown zone, the pre-existing plant community disappeared, and flood-resistant plants such as Salix subfragilis increased. The natatorial bird population continued to grow for 4 years after dam reservoir emergence and stabilised thereafter. Every year, the majority of natatorial birds utilising the dam reservoir as a resting area were ducks, but populations of diving ducks fluctuated depending on water level and iced area. After impoundment, the fish populations increased. As in most dam reservoirs in Japan, populations of invasive fish species such as Micropterus salmoides and Lepomis macrochirus increased. However, spawning grounds dried up during low-water-level seasons, suggesting that regulating water levels may help reduce invasive species.     

Streptococcal preparation OK-432 promotes fusion efficiency and enhances induction of antigen-specific CTL by fusions of dendritic cells and colorectal cancer cells

Dendritic/tumor fusion cell (FC) vaccine is an effective approach for various types of cancer but has not yet been standardized. Antitumor activity can be modulated by different mechanisms such as dendritic cell (DC) maturation state. This study addressed optimal strategies for FC preparations to enhance Ag-specific CTL activity. We have created three types of FC preparations by alternating fusion cell partners: 1) immature DCs fused with autologous colorectal carcinoma cells (Imm-FCs); 2) Imm-FCs followed by stimulation with penicillin-inactivated Streptococcus pyogenes (OK-432) (Imm-FCs/OK); and 3) OK-432-stimulated DCs directly fused to autologous colorectal carcinoma cells (OK-FCs). Both OK-FCs and Imm-FCs/OK coexpressed the CEA, MUC1, and significantly higher levels of CD86, CD83, and IL-12 than those obtained with Imm-FCs. Short-term culture of fusion cell preparations promoted the fusion efficiency. Interestingly, OK-FCs were more efficient in stimulating CD4(+) and CD8(+) T cells capable of high levels of IFN-gamma production and cytolysis of autologous tumor or semiallogeneic targets. Moreover, OK-FCs are more effective inducer of CTL activation compared with Imm-FCs/OK on a per fusion cell basis. The pentameric assay confirmed that CEA- and MUC1-specific CTL was induced simultaneously by OK-FCs at high frequency. Furthermore, the cryopreserved OK-FCs retained stimulatory capacity for inducing antitumor immunity. These results suggest that OK-432 promotes fusion efficiency and induction of Ag-specific CTL by fusion cells. We conclude that DCs fused after stimulation by OK-432 may have the potential applicability to the field of antitumor immunotherapy and may provide a platform for adoptive immunotherapy in the clinical setting.     

Bmi1 cooperates with Dnmt1-associated protein 1 in gene silencing

Polycomb group (PcG) proteins are involved in gene silencing through chromatin modifications. Among polycomb repressive complexes (PRCs), PRC1 exhibits H2A-K119 ubiquitin E3 ligase activity. However, the molecular mechanisms underlying PRC1-mediated gene silencing remain largely obscure. In this study, we found that Bmi1 directly interacts with Dnmt-associated protein 1 (Dmap1), which has been characterized to associate with the maintenance DNA methyltransferase, Dnmt1. Bmi1 was demonstrated to form a ternary complex with Dmap1 and Dnmt1 with Dmap1 in the central position. Chromatin immunoprecipitations confirmed the ternary complex formation within the context of the PRC1 at the Bmi1 target loci. Loss of Dmap1 binding to the Bmi1 target loci was tightly associated with derepressed gene expression in Bmi1-/- cells. Dmap1 knockdown exhibited the same impact as Bmi1 knockout did on the expression of Bmi1 targets, including Hox genes. Collectively, our findings suggest that Bmi1 incorporates Dmap1 in polycomb gene silencing.     

Early downregulation of IGF-I decides the fate of rat retinal ganglion cells after optic nerve injury

Retinal ganglion cells (RGCs) die by apoptosis after optic nerve injury. A number of reports have separately shown changes in pro-apoptotic proteins such as the Bcl-2 family members following optic nerve injury. However, induction time of these apoptotic signals has not been identified due to different treatments of the optic nerve, and insufficient time intervals for measurements. Therefore, the stream of cell death signals is not well understood. In the present study, we systematically reinvestigated a detailed time course of these cell death/survival signals in the rat retina after optic nerve crush, to determine the signal cascade leading to RGC apoptosis. The most conspicuous changes detected in the retina were the rapid inactivation of phospho-Akt and phospho-Bad proteins 2-3 days after optic nerve damage, and the subsequent gradual activation of Bax protein and caspase-3 activity accompanied by cell loss of RGCs 6 days after nerve injury. Cellular localization of these molecular changes was limited to RGCs. Furthermore, amount of insulin-like growth factor-I (IGF-I), an activator of the phosphatidyl inositol-3-kinase (PI3K)/Akt system, was initially decreased from RGCs 1-2 days just prior to the inactivation of phospho-Akt by optic nerve crush. Conversely, supplementation with IGF-I into the rat retina induced upregulation of phospho-Akt expression and cell survival of RGCs both in vitro and in vivo. Thus, injury to the optic nerve might induce early changes in cellular homeostasis with a plausible loss of trophic support for injured RGCs. Actually, IGF-I drastically enhanced neurite outgrowth from adult rat RGCs via a wortmannin-dependent mechanism in a retinal explant culture. Our data strongly indicate that IGF-I is a key molecule that induces RGC apoptosis or RGC survival and regeneration in the retina during the early stage of optic nerve injury.     

Monte Carlo-based calculation of imaging plate response to <Superscript>90</Superscript>Sr in teeth: experimental validation of the required correction on sample thickness

Recently, a numerical method was proposed to correct the imaging plate (IP) response to ⁹⁰Sr concentration in tooth samples, depending on the sample thickness. This is important to quantify any ⁹⁰Sr concentration in teeth, which in turn is necessary to determine any ⁹⁰Sr incorporation of a person retrospectively. Although the final goal will be to evaluate the (inhomogeneous) spatial distribution of ⁹⁰Sr inside tooth samples precisely, the present study was restricted—as a first step—to the evaluation of ⁹⁰Sr in teeth assuming a uniform ⁹⁰Sr distribution. A numerical method proposed earlier was validated experimentally in the present study by measuring the IP response to standard sources of various thicknesses and ⁹⁰Sr concentrations. For comparison, the energy deposition of the β-rays emitted by ⁹⁰Sr in the IP—which is considered to be proportional to the IP luminescence signal—was calculated for the various sample thicknesses involved, by means of the MCNP-4C code. As a result, the measured IP response could be reproduced by the calculations within the uncertainties, depending on the thickness of the standard sources. Thus, the validity of the proposed numerical method to correct the IP response for sample thickness has successfully been demonstrated.