Role of the GH-IGF-1 system in Atlantic salmon and rainbow trout postsmolts at elevated water temperature.

A comparative experiment with Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) postsmolts was conducted over 35 days to provide insight into how growth, respiration, energy metabolism and the growth hormone (GH) and insulin-like growth factor 1 (IGF-1) system are regulated at elevated sea temperatures. Rainbow trout grew better than Atlantic salmon, and did not show reduced growth at 19 °C. Rainbow trout kept at 19 °C had increased blood hemoglobin concentration compared to rainbow trout kept at 13 °C, while salmon did not show the same hemoglobin response due to increased temperature. Both species showed reduced length growth and decreased muscle glycogen stores at 19 °C. Circulating IGF-1 concentration was higher in rainbow trout than in Atlantic salmon, but was not affected by temperature in either species. Plasma IGF-binding protein 1b (IGFBP-1b) concentration was reduced in Atlantic salmon reared at 19 °C after 15 days but increased in rainbow trout at 19 °C after 35 days. The igfbp1b mRNA level in liver showed a positive correlation to plasma concentrations of glucose and IGFBP-1b, suggesting involvement of this binding protein in carbohydrate metabolism at 19 °C. At this temperature muscle igfbp1a mRNA was down-regulated in both species. The muscle expression of this binding protein correlated negatively with muscle igf1 and length growth. The plasma IGFBP-1b concentration and igfbp1b and igfbp1a expression suggests reduced muscle igf1 signaling at elevated temperature leading to glucose allostasis, and that time course is species specific due to higher thermal tolerance in rainbow trout.

Development of a time-resolved fluoroimmunoassay for salmon insulin-like growth factor binding protein-1b.

In salmon plasma/serum, three major insulin-like growth factor binding proteins (IGFBPs) are consistently detected at 22-, 28- and 41-kDa. The 22-kDa form has been identified as IGFBP-1b and shown to increase under catabolic conditions. We developed a competitive time-resolved fluoroimmunoassay (TR-FIA) for salmon IGFBP-1b. Purified salmon IGFBP-1b was used for biotin-labeling, assay standard and antiserum production. The TR-FIA did not cross-react with the 41-kDa form (IGFBP-2b) but showed 3% cross-reactivity with the 28-kDa form (IGFBP-1a). It measured IGFBP-1b levels as low as 0.4 ng/ml, and ED80 and ED20 were 0.9 and 24.6 ng/ml, respectively. There appears to be little interference by IGF-I. Using the TR-FIA, serum IGFBP-1b levels were measured in individually-tagged underyearling masu salmon fed or fasted for 5 weeks, or fasted for 3 weeks followed by refeeding for 2 weeks. Fasting for 3 weeks significantly increased circulating IGFBP-1b levels, while it returned to the basal levels after prolonged fasting for additional 2 weeks. Serum IGFBP-1b level negatively correlated with body weight, condition factor, specific growth rate and serum IGF-I level. During parr-smolt transformation of masu salmon, average circulating IGFBP-1b levels were the highest in May. There was a positive correlation between serum IGFBP-1b and IGF-I, which is in contrast to that in the fasting/feeding experiment. IGFBP-1b also showed a positive relationship with gill Na(+), K(+)-ATPase activity. These results suggest that the relationship between circulating IGFBP-1b and IGF-I during smoltification differs from that during fasting and IGFBP-1b may play a role in the development of hypoosmoregulatory ability.

Development of a novel protocol for generating flavivirus reporter particles.

Infection with West Nile virus (WNV), a mosquito-borne flavivirus, is a growing public and animal health concern worldwide. Prevention, diagnosis and treatment strategies for the infection are urgently required. Recently, viral reverse genetic systems have been developed and applied to clinical WNV virology. We developed a protocol for generating reporter virus particles (RVPs) of WNV with the aim of overcoming two major problems associated with conventional protocols, the difficulty in generating RVPs due to the specific skills required for handling RNAs, and the potential for environmental contamination by antibiotic-resistant genes encoded within the genome RNA of the RVPs. By using the proposed protocol, cells were established in which the RVP genome RNA is replicated constitutively and does not encode any antibiotic-resistant genes, and used as the cell supply for RVP genome RNA. Generation of the WNV RVPs requires only the simple transfection of the expression vectors for the viral structural proteins into the cells. Therefore, no RNA handling is required in this protocol. The WNV RVP yield obtained using this protocol was similar that obtained using the conventional protocol. According to these results, the newly developed protocol appears to be a good alternative for the generation of WNV RVPs, particularly for clinical applications.

Responses of insulin-like growth factor (IGF)-I and two IGF-binding protein-1 subtypes to fasting and re-feeding, and their relationships with individual growth rates in yearling masu salmon (Oncorhynchus masou).

Two subtypes of insulin-like growth factor binding protein (IGFBP)-1 are present in salmon blood and they are both up-regulated under catabolic conditions such as stress. The present study examined effects of fasting and re-feeding on IGFBP-1a (28-kDa form) and IGFBP-1b (22-kDa form) both at mRNA and protein levels along with IGF-I and RNA/DNA ratio in yearling masu salmon. Fish were individually tagged and assigned to one of three treatments: Fed, Fasted or Re-fed. Circulating IGF-I levels significantly decreased after fasting for 5 weeks and were positively correlated with individual growth rates. Liver igf-1 mRNA levels were not affected by the treatment. Muscle RNA/DNA ratio did not respond to fasting nor showed correlations with growth rates. Circulating IGFBP-1a and IGFBP-1b increased during fasting and decreased after re-feeding. Both serum levels were inversely correlated with growth rates, while IGFBP-1b had consistent negative relationships with growth rates. Fasting/re-feeding also affected their mRNA levels in the liver. These results suggest that circulating IGF-I and IGFBP-1b could serve as positive and negative indices of growth, respectively, in masu salmon. Different sensitivities of IGBP-1a and IGFBP-1b may be useful to assess a broad range of catabolic conditions when they are combined.

Jungle honey enhances immune function and antitumor activity.

Jungle honey (JH) is collected from timber and blossom by wild honey bees that live in the tropical forest of Nigeria. JH is used as a traditional medicine for colds, skin inflammation and burn wounds as well as general health care. However, the effects of JH on immune functions are not clearly known. Therefore, we investigated the effects of JH on immune functions and antitumor activity in mice. Female C57BL/6 mice were injected with JH (1 mg/mouse/day, seven times intra-peritoneal). After seven injections, peritoneal cells (PC) were obtained. Antitumor activity was assessed by growth of Lewis Lung Carcinoma/2 (LL/2) cells. PC numbers were increased in JH-injected mice compared to control mice. In Dot Plot analysis by FACS, a new cell population appeared in JH-injected mice. The percent of Gr-1 surface antigen and the intensity of Gr-1 antigen expression of PC were increased in JH-injected mice. The new cell population was neutrophils. JH possessed chemotactic activity for neutrophils. Tumor incidence and weight were decreased in JH-injected mice. The ratio of reactive oxygen species (ROS) producing cells was increased in JH-injected mice. The effective component in JH was fractionized by gel filtration using HPLC and had an approximate molecular weight (MW) of 261. These results suggest that neutrophils induced by JH possess potent antitumor activity mediated by ROS and the effective immune component of JH is substrate of MW 261.

1-(3'-[125I]Iodophenyl)-3-methy-2-pyrazolin-5-one: preparation, solution stability, and biodistribution in normal mice.

3-Methyl-1-phenyl-2-pyrazolin-5-one (edaravone, 1), known as a potent free radical scavenger, has been developed as a medical drug for the treatment of acute cerebral infarction. With the aim of developing radiotracers for imaging free radicals in vivo, 1-(3'-[(125)I]iodophenyl)-3-methy-2-pyrazolin-5-one ((125)I-2) was synthesized by two methods, via isotopic exchange and interhalogen exchange under solvent-free conditions, in which iodo- and bromo-derivatives were used as labeling precursors, respectively. After HPLC purification, (125)I-2 was obtained in modest isolated radiochemical yields (ca. 20%) with high radiochemical purities by both methods. The former gave specific activities of 0.2-0.6 kBq/micromol, whereas the latter approach achieved specific activities of more than 0.14 GBq/micromol. On attempting to prepare an injectable formulation for (125)I-2 with high specific activity, its radiochemical purities dropped to about 60-70%. Unlabeled analog 2 was found to have lipophilic and antioxidant properties similar to edaravone. Intravenous injection of (125)I-2 with low specific radioactivity into normal mice showed signs of distribution profiles similar to reported results for (14)C-labeled edaravone in normal rats.

New approaches to mechanism analysis for drug discovery using DNA microarray data combined with KeyMolnet.

We have developed a comprehensive information platform, named KeyMolnet, for drug discovery and life science research in the post-genome era. Using KeyMolnet, we show new approaches to research into the biological mechanism in DNA microarray analysis. Thanks to the DNA microarray technology, it is now possible to obtain very large quantities of gene expression data at a time. However, it is still difficult to extract meaningful information from such large quantities of data and to analyze the relationship between gene expression data and biological function. We therefore developed an advanced tool that can generate molecular networks upon demand, and beyond signaling "cross-talks," can connect them to physiological phenomena and medical and drug information. Here we show the methods of mechanism analysis using the DNA microarray data and KeyMolnet, as well as the possible mechanism of inducing apoptosis in the human promyelocytic leukemia cell line, HL-60, treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), using the time series of gene expression data from DNA microarray experiments. KeyMolnet enables practical approaches to research into biological mechanisms, which in turn contribute to new discoveries in the medical, pharmaceutical and life sciences.

Increased interleukin-1 and interleukin-1 receptor antagonist levels in cervical mucus in the ovulatory phase in comparison with the follicular phase.

Interleukin-1 (IL-1) receptor antagonist (IL-1ra) levels in the cervical mucus of women in the ovulatory phase are significantly higher than those in the follicular phase. IL-1 titers of women in the ovulatory phase are also significantly higher than those in the follicular phase. A positive correlation between IL-1ra and IL-1 levels in the cervical mucus was observed. Immunohistochemistry using an anti-IL-1ra monoclonal antibody revealed positive staining in the epithelial cells of the endocervix. These results suggest that IL-1ra from cervical epithelial cells protects the reproductive system from the toxicity of IL-1 produced in the endocervix.