Four domains of Ada1 form a heterochromatin boundary through different mechanisms.

In eukaryotic cells, there are two chromatin states, silenced and active, and the formation of a so-called boundary plays a critical role in demarcating these regions; however, the mechanisms underlying boundary formation are not well understood. In this study, we focused on S. cerevisiae ADA1, a gene previously shown to encode a protein with a robust boundary function. Ada1 is a component of the histone modification complex Spt-Ada-Gcn5-acetyltransferase (SAGA) and the SAGA-like (SLIK) complex, and it helps to maintain the integrity of these complexes. Domain analysis showed that four relatively small regions of Ada1 (Region I; 66-75 aa, II; 232-282 aa, III; 416-436 aa and IV; 476-488 aa) have a boundary function. Among these, Region II could form an intact SAGA complex, whereas the other regions could not. Investigation of cellular factors that interact with these small regions identified a number of proteasome-associated proteins. Interestingly, the boundary functions of Region II and Region III were affected by depletion of Ump1, a maturation and assembly factor of the 20S proteasome. These results suggest that the boundary function of Ada1 is functionally linked to proteasome processes and that the four relatively small regions in ADA1 form a boundary via different mechanisms.

C-terminus of the Sgf73 subunit of SAGA and SLIK is important for retention in the larger complex and for heterochromatin boundary function.

The budding yeast Saccharomyces cerevisiae contains active and inactive chromatin separated by boundary domains. Previously, we used genome-wide screening to identify 55 boundary-related genes. Here, we focus on Sgf73, a boundary protein that is a component of the Spt-Ada-Gcn5 acetyltransferase (SAGA) and SLIK (SAGA-like) complexes. These complexes have histone acetyltransferase (HAT) and histone deubiquitinase activity, and Sgf73 is one of the factors necessary to anchor the deubiquitination module. Domain analysis of Sgf73 was carried out, and the minimum region (373-402 aa) essential for boundary function was identified. This minimum region does not include the domain involved in anchoring the deubiquitination module, suggesting that the histone deubiquitinase activity of Sgf73 is not important for its boundary function. Next, Sgf73-mediated boundary function was analyzed in disruption strains in which different protein subunits of the SAGA/SLIK/ADA complexes were deleted. Deletion of ada2, ada3 or gcn5 (a HAT module component) caused complete loss of the boundary function of Sgf73. The importance of SAGA or SLIK complex binding to the boundary function of Sgf73 was also analyzed. Western blot analysis detected both the full-length and truncated forms of Spt7, suggesting that SAGA and SLIK complex formation is important for the boundary function of Sgf73.

Regulation of gene expression in retrovirus vectors by X-ray and proton beam radiation with artificially constructed promoters.

BACKGROUND: We previously obtained an X-ray responsive promoter from 11 promoters that we constructed. In the present study, we aimed to determine the efficiency of our promoter construction method. In addition, the reactivity of the promoter to X-rays in vivo is also investigated. METHODS: Promoters constructed by linking the TATA box to randomly combined binding sequences of transcription factors activated by radiation were cloned to prepare a promoter library. Combinations of promoters and various genes were stably-transfected into HeLa cells to establish recombinant cell lines, which were then exposed to X-rays or a proton beam to observe gene expression enhancement with or without anti-oxidants. Tumors of luciferase-expressing recombinant cells on mice were exposed to X-rays and promoter activation was evaluated by detecting bioluminescence. As a model for in vitro suicide gene therapy, fcy::fur-expressing recombinant cells were exposed to X-rays before incubation with 5-fluorocytosin. Cell viability was determined with WST-8. RESULTS: Twenty-five of the 62 promoters in the library enhanced luciferase activity over five-fold, 6 h after receiving 10 Gy of X-ray irradiation, suggesting the effectiveness of our method. Luciferase activity in recombinant cells was enhanced by X-rays and, to a lesser extent, by a proton beam. Anti-oxidants attenuated the enhancement, suggesting the involvement of oxidative stress. Promoters were less reactive to X-rays in tumors on mice. In our suicide gene therapy model, survival of post-irradiated cells decreased dose-dependently with 5-fluorocytosin. CONCLUSIONS: Our method was efficient in generating radiation responsive promoters. Furthermore, we have successfully shown a potential therapeutic use for one of these promoters.

Real-time visualization of intratumoral necrosis using split-luciferase reconstitution by protein trans-splicing.

Necrosis, a form of cell death, occurs not only with the development of various diseases but also with a tumor tissue response to cancer treatment. Therefore, pursuing progress for cancer therapy through induction of necrosis may be one of the most effective approaches for cancer eradication. We herein describe the development of a real-time imaging system to visualize intratumoral necrosis. The system is composed of two types of cells expressing either one of two necrosis imaging reporters that consist of a DnaE intein sequence linking to one of two split-luciferase fragments. When necrosis occurs in a tumor composed of both of the cells, the two types of leaked reporters can reconstitute the enzymatic activity as a result of protein trans-splicing and thereby emit bioluminescence in the presence of the substrate. This system, which was constructed with shrimp-derived luciferase, allowed in vitro imaging of necrosis. We further confirmed real-time imaging of intratumoral necrosis caused by physical or chemical tissue disruption, validating its application in in vivo necrosis imaging. Thus, the constructed imaging system could be a powerful tool for the optimization of the therapeutic condition for cancer therapy and for the evaluation of novel anticancer drugs targeting necrosis.

A new paradigm in radioadaptive response developing from microbeam research.

A classic paradigm in radiation biology asserts that all radiation effects on cells, tissues and organisms are due to the direct action of radiation on living tissue. Using this model, possible risks from exposure to low dose ionizing radiation (below 100 mSv) are estimated by extrapolating from data obtained after exposure to higher doses of radiation, using a linear non-threshold model (LNT model). However, the validity of using this dose-response model is controversial because evidence accumulated over the past decade has indicated that living organisms, including humans, respond differently to low dose/low dose-rate radiation than they do to high dose/high dose-rate radiation. These important responses to low dose/low dose-rate radiation are the radiation-induced adaptive response, the bystander response, low-dose hypersensitivity, and genomic instability. The mechanisms underlying these responses often involve biochemical and molecular signals generated in response to targeted and non-targeted events. In order to define and understand the bystander response to provide a basis for the understanding of non-targeted events and to elucidate the mechanisms involved, recent sophisticated research has been conducted with X-ray microbeams and charged heavy particle microbeams, and these studies have produced many new observations. Based on these observations, associations have been suggested to exist between the radioadaptive and bystander responses. The present review focuses on these two phenomena, and summarizes observations supporting their existence, and discusses the linkage between them in light of recent results obtained from experiments utilizing microbeams.

Microbeam irradiation facilities for radiobiology in Japan and China.

In order to study the radiobiological effects of low dose radiation, microbeam irradiation facilities have been developed in the world. This type of facilities now becomes an essential tool for studying bystander effects and relating signaling phenomena in cells or tissues. This review introduces you available microbeam facilities in Japan and in China, to promote radiobiology using microbeam probe and to encourage collaborative research between radiobiologists interested in using microbeam in Japan and in China.

Thermosensitization by parthenolide in human lung adenocarcinoma A549 cells and p53- and hsp72-independent apoptosis induction via the nuclear factor-kappaB signal pathway.

The thermo-enhancement effects of the sesquiterpene lactone parthenolide (PTL), which targets the transcription factor nuclear factor-kappaB (NF-kappaB), and hyperthermia at 40, 42 and 44 degrees C on the human lung adenocarcinoma A549 cell line were investigated in vitro. Thermotherapy using a combined treatment with PTL (0.02 microM) prior to hyperthermia showed synergistic thermo-enhancement effects towards A549 cells. The expression of p53 and hsp72 proteins following the application of PTL and hyperthermia at 44 degrees C, both alone and in combination, were examined to investigate whether p53 and hsp72 participated in apoptosis induction via the NF-kappaB signal pathway. After treatment with PTL alone, Hsp72 was only slightly induced, which was the same as for the control, while the level following the combination treatment was not significantly different as compared with hyperthermia alone. In addition, the level of p53 after the combination treatment was only slightly increased in comparison with hyperthermia alone. The kinetics of apoptosis and necrosis induction during the incubation periods following PTL exposure and hyperthermia, and the combination of both were also determined. The incidence of apoptosis following hyperthermia alone was approximately 0.6% on average after 12, 24 and 48 h of incubation, while that of PTL alone was approximately 1.7%, and that with the combination treatment was around 2.3%. Thus, induction of apoptosis following the combination treatment was increased as compared to each treatment alone. With regard to the kinetics of necrosis, the incidence of necrosis after treatment with hyperthermia alone was approximately 2.7%, while that with the combination treatment was lower, at around 2.2%. We hypothesized that cells treated with PTL had an altered arrangement of stressed cells undergoing the transformation from necrotic cell death to apoptotic cell death via another mechanism. Our results suggested that the PTL-induced apoptosis of A549 cells was due to the direct suppression of NF-kappaB activity in a p53- and hsp72-independent manner based on NF-kappaB signaling.

Enhancement of radiosensitivity by topoisomerase II inhibitor, amrubicin and amrubicinol, in human lung adenocarcinoma A549 cells and kinetics of apoptosis and necrosis induction.

The effects of amrubicin (AMR) and its active metabolite, amrubicinol (AMROH), on the sensitivity of human lung adenocarcinoma A549 cells to ionizing radiation were investigated in vitro. Further, the kinetics of apoptosis and necrosis induction were also analyzed. The cytocidal effects of X-ray irradiation on A549 cells resulted in a low level of radiosensitivity with a D0 value of 12 Gy. The slopes of the survival curves in the exponential phase were plotted on semilogarithmic paper for radiation combined with AMR (2.5 microg/ml) and AMROH (0.02 microg/ml) treatment, and were shown to be approximately parallel to treatment with irradiation alone. The initial shoulder-shape portion of the survival curve for radiation alone, indicating the repair of sublethal damage, was reduced as compared to that for sequential combined treatment with AMR or AMROH. Sequential treatments with AMR or AMROH prior to ionizing radiation resulted in an additive radio-enhancement effect that reduced not only survival, but also the shoulder width. Fractionated irradiation with 2 Gy per fraction of A549 cells was carried out in vitro similar to that commonly performed in clinical radiotherapy and the radio-resistance of the cells was shown to be inhibited by AMR and AMROH. Similar to AMR and AMROH, adriamycin and etoposide (VP-16) are DNA topoisomerase II inhibitors. The effects of these 4 agents on cells that received X-ray irradiation were compared and all of the agents exhibited comparable radio-enhancement effects. The induction of apoptosis was investigated at 48 and 72 h after administration of AMROH, radiation or combined treatment, and apoptosis was not significantly induced after any of the treatments. We also examined the induction of necrosis, and found that the incidence of necrosis following combined treatment was approximately 2 times higher than that with either of the single treatments.

Development of a real-time imaging system for hypoxic cell apoptosis.

Hypoxic regions within the tumor form due to imbalances between cell proliferation and angiogenesis; specifically, temporary closure or a reduced flow due to abnormal vasculature. They create environments where cancer cells acquire resistance to therapies. Therefore, the development of therapeutic approaches targeting the hypoxic cells is one of the most crucial challenges for cancer regression. Screening potential candidates for effective diagnostic modalities even under a hypoxic environment would be an important first step. In this study, we describe the development of a real-time imaging system to monitor hypoxic cell apoptosis for such screening. The imaging system is composed of a cyclic luciferase (luc) gene under the control of an improved hypoxic-responsive promoter. The cyclic luc gene product works as a caspase-3 (cas-3) monitor as it gains luc activity in response to cas-3 activation. The promoter composed of six hypoxic responsible elements and the CMV IE1 core promoter drives the effective expression of the cyclic luc gene in hypoxic conditions, enhancing hypoxic cell apoptosis visualization. We also confirmed real-time imaging of hypoxic cell apoptosis in the spheroid, which shares properties with the tumor. Thus, this constructed system could be a powerful tool for the development of effective anticancer diagnostic modalities.

Generation of a strong promoter for Escherichia coli from eukaryotic genome DNA.

Improvement of a gene product by introducing mutations into the gene is usually applied for improving structural genes. In this study the procedure was applied for generation and improvement of a genetic signal to drive gene expression. By adding various concentrations of Mn2+ to the PCR reaction mixture, mutations were introduced into a DNA fragment at various ratios. An appropriate condition was employed to introduce mutations into a DNA fragment with no promoter activity. The mutated fragment was introduced at an upstream site of the lacZ gene in a plasmid vector to see if the fragment carries promoter activity. Lysate of an Escherichia coli transformant with the vector was assayed for beta-galactosidase expression as an indicator of the promoter activity. Mutated DNA fragments were generated by error prone PCR with a condition which leads to introduction of 1.5% of mutation into a DNA fragment during the process. The strongest promoter was chosen by beta-galactosidase assay after error prone PCR and subjected to another step of the PCR. These processes were repeated four times to improve its activity to 1.94-fold to that by the tac promoter. When the luciferase gene was expressed by the strongest promoters, a similar expression level was noted. These results indicate that by randomly introducing mutations into a DNA fragment, it is relatively easy to generate and improve a prokaryotic promoter.

Modification of thermosensitivity by amrubicin or amrubicinol in human lung adenocarcinoma A549 cells and the kinetics of apoptosis and necrosis induction.

The effects of amrubicin (AMR) and its active metabolite, amrubicinol (AMROH), on the sensitivity of human lung adenocarcinoma A549 cell line to hyperthermia at 44 degrees C were investigated. The cell phase response as well as the kinetics of apoptosis and necrosis induction were also analyzed. The cytocidal effects of 44 degrees C hyperthermia on A549 cells exhibited low thermosensitivity with a T(0) value of 12 min. The slope of the survival curve in the exponential phase, described semilogarithmically, in 44 degrees C hyperthermia combined treatment with AMROH (0.02 microg/ml) was approximately parallel to 44 degrees C hyperthermia alone. The initial shoulder shape portion of the survival curve from 44 degrees C hyperthermia alone, indicating the repair of sublethal thermal damage (SLTDR), was reduced with the sequential combined treatment of AMR or AMROH. Sequential treatments with AMR or AMROH prior to 44 degrees C hyperthermia resulted in additive thermo-enhancement effect by reducing not only survival but was shoulder wide. Furthermore, like AMR and AMROH, adriamycin (ADM) and etoposide (VP-16) are DNA topoisomerase II inhibitors, and the effects of these 4 agents on 44 degrees C hyperthermia were compared. All 4 agents exhibited comparable thermo-enhancement effects. Using synchronized A549 cells, AMR or AMROH did not elicit cell phase responses, irrespective of the concentration. The induction of apoptosis was investigated at 48 and 72 h after AMROH treatment, 44 degrees C hyperthermia or the combined treatment, in which apoptosis was not significantly induced after any treatment. Furthermore, the incidence of necrosis was examined as well as apoptosis. The incidence of necrosis at 48 and 72 h after AMROH was 2.4 and 4.3%, respectively; after 44 degrees C hyperthermia was 3.3 and 4.0%, respectively; and after the combined treatment it was 10.7 and 8.7%, respectively. The necrosis induced after the combined treatment was circa 3 times higher than that in either of the single treatments.

Simple and efficient isolation of cordycepin from culture broth of a Cordyceps militaris mutant.

Isolation of cordycepin from the culture broth of Cordyceps militaris mutant was investigated. Based on the solubility curve, three crystallizing processes, temperature shift (process I), pH shift (process II), and pH shift followed by temperature shift (process III) were carried out. Process III was the most promising method regarding both purity and yield.

Selective enhancement of hypoxic cell killing by tempol-regulated suicide gene expression.

The presence of hypoxic regions within solid tumors is caused by an imbalance between cell proliferation and angiogenesis. Such regions may facilitate the onset of recurrence after radiation therapy and chemotherapy, as hypoxic cells show resistance to these treatments. We found that tempol, a nitroxide, strongly induces the accumulation of hypoxia-inducible factor (HIF)-1α, particularly under conditions of hypoxia. We, therefore, evaluated whether tempol enhances the gene expression via HIF-1α, potentially leading to various applications for cancer gene therapy targeting hypoxic cells. Consequently, following treatment with tempol under hypoxia, the luciferase (Luc) activity in the cells transfected with the plasmid containing the luc gene with the oxygen-dependent degradation domain and a promoter composed of hypoxia-responsive elements increased up to approximately 10-fold compared to that observed in cells treated identically with the exception of tempol. The plasmid constructed by replacing the luc gene with the fcy::fur fusion gene as a suicide gene, strongly induced the accumulation of the Fcy::Fur fusion protein, only when incubated in the presence of the hypoxic mimic CoCl2 and tempol. The transfected cells were successfully killed with the addition of 5-fluorocytosine to the cell culture according to the fcy::fur fusion gene expression. As similar but lesser enhancement of the Luc activity was also observed in solid tumor tissues in nude mice, this strategy may be applied for hypoxic cancer eradication.

P53-independent thermosensitization by mitomycin C in human non-small-cell lung cancer cells.

PURPOSE: To elucidate the relationship between p53 functions and the interactive effects of the combined treatment with mild hyperthermia and mitomycin C. METHODS AND MATERIALS: p53-deficient human non-small-cell lung cancer H1299 cells were transfected with a vector carrying a neomycin-resistant gene (neo) or together with a wild-type or mutant p53 gene. Sensitivities of these transfectants to mild hyperthermia at 42 degrees C, mitomycin C (0.05 microg/mL) at 37 degrees C, or the combination treatment were determined by colony formation assay. After these treatments, the induction of apoptosis, the changes in cell cycle distribution, and the accumulation of Hsp72 were examined. RESULTS: The combined treatment resulted in an enhanced cell killing effect in H1299 cells in a p53-independent manner, which was partially the result of an enhancement of heat-induced apoptosis. The treatment also caused a marked G(2)/M arrest in the neo and the mutant p53 cells, but not in the wild-type p53 cells. The subsequent release of G(2)/M arrest was accompanied with an increase in the sub-G(1) fractions. Mitomycin C did not affect the accumulation of Hsp72 induced by hyperthermia in H1299 cells regardless of their p53 gene status. CONCLUSION: Our findings demonstrate a p53-independent mechanism for an interactively cytotoxic enhancement by combined treatment with mild hyperthermia and mitomycin C.

Effects of combined treatment with 40 degrees C hyperthermia and bleomycin on the accumulation of heat shock protein in murine L cells.

Our research group has reported the enhanced cytotoxicity of combined treatment with bleomycin (BLM) and low hyperthermia at 40 degrees C, using murine L cells, and suggested that post-heating could inhibit BLM-induced sublethal damage repair. For further understanding of the involved mechanisms, we subsequently investigated the kinetics of the cellular accumulations of inducible 72-kDa heat shock protein (hsp72) after 40 degrees C hyperthermia and/or BLM treatment using the same cell line. Western blot analysis showed significantly enhanced accumulation of hsp72 after a low hyperthermia at 40 degrees C for 40, 105 or 180 min, and no significant enhancement of it after exposure to 10 microg/ml BLM at 37 degrees C for either 40 or 105 min. When the cells were heated in the presence of BLM, the accumulations of hsp72 were markedly suppressed, with the maxima of hsp72 accumulation decreasing to 38% and 63% of those induced by hyperthermia alone for 40 or 105 min, respectively. On the other hand, sequential treatment with hyperthermia either before or after BLM treatment did not show significant alteration of the heat-induced accumulations of hsp72. It was demonstrated that BLM was necessary during heating to effectively suppress the heat-induced accumulation of hsp72. This study indicates that the suppression of heat-induced accumulation of hsp72 by BLM may partially contribute to enhance cytotoxicity of the simultaneous treatment of 40 degrees C hyperthermia and BLM.

Sinodielide A exerts thermosensitizing effects and induces apoptosis and G2/M cell cycle arrest in DU145 human prostate cancer cells via the Ras/Raf/MAPK and PI3K/Akt signaling pathways.

Sinodielide A (SA) is a naturally occurring guaianolide, which is isolated from the root of Sinodielsia yunnanensis. This root, commonly found in Yunnan province, is used in traditional Chinese medicine as an antipyretic, analgesic and diaphoretic agent. A number of studies have reported that agents isolated from a species of Umbelliferae (Apiaceae) have antitumor activities. We previously reported, using combined treatments with this medicinal herb and hyperthermia at various temperatures, an enhanced cytotoxicity in the human prostate cancer androgen­independent cell lines, PC3 and DU145, and analyzed the related mechanisms. In the present study, we investigated the effects of treatment with SA prior to hyperthermia on the thermosensitivity of DU145 cells, and the mechanisms related to the induction of apoptosis and G(2)/M cell cycle arrest via the activation of extracellular-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) signaling pathways, as well as the phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. Cells were exposed to hyperthermia alone (40-44˚C) or hyperthermia in combination with SA. Lethal damage to cells treated with mild hyperthermia (40 or 42˚C) for up to 6 h was slight; however, hyperthermia in combination with SA synergistically enhanced thermosensivity. Lethal damage to cells treated with acute hyperthermia (43 or 44˚C) was more severe, but these effects were also enhanced and were more significant by the combined treatment with SA. The kinetics of apoptosis induction and cell cycle distribution were analyzed by flow cytometry. In addition, the levels of ERK1/2, JNK and Akt were determined by western blot analysis. The incidence of apoptotic cells after treatment with SA (20.0 µM) at 37˚C for 4 h, hyperthermia (44˚C) alone for 30 min, and the combination in sequence were examined. The sub-G1 division (%) in the diagram obtained by flow cytometry was applied to that assay. The percentage of apoptotic cells (10.53±5.02%) was higher at 48 h as compared to 0, 12 and 24 h after treatment. The distribution of DU145 cells in the G2/M cell cycle phase was markedly increased after 24 h of heating at 44˚C and after the combined treatment with heating and SA. The phosphorylation of ERK1/2 was reduced following treatment with heating and SA, while the levels of phosphorylated JNK (p-JNK) were markedly increased immediately after heating at 44˚C and when heating was combined with SA. By contrast, the levels of phosphorylated Akt (p-Akt) were immediately increased only after heating at 44˚C. Thus, we concluded that SA exerts its thermosensitizing effects on DU145 cells by inhibiting the activation of the MAPK/ERK1/2 and PI3K/Akt signaling pathways.

Relative biological effectiveness of therapeutic proton beams for HSG cells at Japanese proton therapy facilities.

We investigated the relative biological effectiveness (RBE) of therapeutic proton beams at six proton facilities in Japan with respect to cell lethality of HSG cells. The RBE of treatments could be determined from experimental data. For this purpose, we used a cell survival assay to compare the cell-killing efficiency of proton beams. Among the five linear accelerator (LINAC) X-ray machines at 4 or 6 MeV that were used as reference beams, there was only a small variation (coefficient of variation CV = 3.1% at D10) in biological effectiveness. The averaged value of D10 for the proton beams at the middle position of the spread-out Bragg peak (SOBP) was 4.98. These values showed good agreement, with a CV of 4.3% among the facilities. Thus, the average RBE10 (RBE at the D10 level) at the middle position of the SOBP beam for six facilities in Japan was 1.05 with a CV of 2.8%.

Development of a therapeutically important radiation induced promoter.

Radio-genetic therapy is a combination of radiation therapy and gene therapy that may solve some of the problems associated with conventional radiotherapy. A promoter responsive to radiation was obtained from a promoter library composed of DNA fragments created by linking the TATA box signal to randomly combined binding sequences of transcription factors that are reactive to radiation. Each promoter connected to the luciferase gene, was evaluated by luciferase expression enhancement in transfected cells after X-ray irradiation. The reactivity of the best promoter was improved by the random introduction of point mutations and the resultant promoter showed more than a 20-fold enhancement of the luciferase expression after X-ray irradiation at 10 Gy. The expression of downstream genes was also enhanced in stably transfected cells not only by X-rays but also by proton beam irradiation; and either enhancement was attenuated when an anti-oxidant was added, thus suggesting the involvement of oxidative stress in the promoter activation. Constructed promoters were also activated in tumors grown in mice. In addition, cell killing with the fcy::fur gene (a suicide gene converting 5-fluorocytosin to highly toxic 5-fluorouracil) increased dose-dependently with 5-fluorocytosin only after X-ray irradiation in vitro. These results suggest that promoters obtained through this method could be used for possible clinical applications.

Production of cordycepin by a repeated batch culture of a Cordyceps militaris mutant obtained by proton beam irradiation.

Cordycepin (3'-deoxyadenosine) is one of the most versatile metabolites of Cordyceps militaris due to its broad spectrum of biological activity. In our previous study, the C. militaris mutant G81-3, which produces higher levels of cordycepin, was obtained by high-energy proton beam irradiation. In this study, the effects of adenosine on cordycepin production in a surface liquid culture of the mutant and the wild type strains were investigated. For the mutant strain, the optimum dose of adenosine yielded a 30% increase in cordycepin production; the maximum levels of production with adenosine and without adenosine were 8.6g/l and 6.7 g/l, respectively. In contrast, the increase due to adenosine supplementation for the wild type strain was only 15% (3.1g/l with adenosine and 2.7 g/l without adenosine). Furthermore, a repeated batch culture, an efficient production method, was carried out to eliminate the relatively long lag phase of the mutant culture. Over four cycles, both the mutant and the wild type strain maintained a production level of more than 85% of that of the initial cycle. As a result, the disadvantage of the mutant was successfully overcome, resulting in a productivity (0.48 g/(ld)) higher than that of the batch culture (0.29 g/(ld)). The productivity for cordycepin obtained in this study is the highest reported value to date, and this method could be applied to large-scale production of cordycepin at industrial levels.

Nitric oxide is a key molecule serving as a bridge between radiation-induced bystander and adaptive responses.

A classical paradigm of radiation biology asserts "targeted effect" that all radiation effects on cells, tissues and organisms are due to the direct action of radiation. However, over the past two decades, a paradigm of radiation biology has undergone a shift away from "targeted effect" relationships and towards complex ongoing "intra- and inter-cellular responses", which involve not only targeted but also non-targeted ones. These responses include now familiar, but still fully unknown, phenomena associated with low-dose/low-dose-rate radiation exposure such as adaptive responses, bystander responses, low-dose hypersensitivity, and genomic instability. The mechanisms underlying these responses often involve biochemical/molecular signals that respond to targeted and non-targeted events. Matsumoto et al. have previously found that nitric oxide functions as initiators of radiation-induced bystander and adaptive responses. These findings suggest correlations between the radioadaptive and bystander responses. The present review focuses on these two phenomena by summarizing observations supporting their existence, and discussing the linkage between them from the aspect of production of reactive nitrogen species.