Computer simulation approach to the identification of visfatin-derived angiogenic peptides.

Angiogenesis plays an essential role in various normal physiological processes, such as embryogenesis, tissue repair, and skin regeneration. Visfatin is a 52 kDa adipokine secreted by various tissues including adipocytes. It stimulates the expression of vascular endothelial growth factor (VEGF) and promotes angiogenesis. However, there are several issues in developing full-length visfatin as a therapeutic drug due to its high molecular weight. Therefore, the purpose of this study was to develop peptides, based on the active site of visfatin, with similar or superior angiogenic activity using computer simulation techniques.Initially, the active site domain (residues 181∼390) of visfatin was first truncated into small peptides using the overlapping technique. Subsequently, the 114 truncated small peptides were then subjected to molecular docking analysis using two docking programs (HADDOCK and GalaxyPepDock) to generate small peptides with the highest affinity for visfatin. Furthermore, molecular dynamics simulations (MD) were conducted to investigate the stability of the protein-ligand complexes by computing root mean square deviation (RSMD) and root mean square fluctuation(RMSF) plots for the visfatin-peptide complexes. Finally, peptides with the highest affinity were examined for angiogenic activities, such as cell migration, invasion, and tubule formation in human umbilical vein endothelial cells (HUVECs). Through the docking analysis of the 114 truncated peptides, we screened nine peptides with a high affinity for visfatin. Of these, we discovered two peptides (peptide-1: LEYKLHDFGY and peptide-2: EYKLHDFGYRGV) with the highest affinity for visfatin. In an in vitrostudy, these two peptides showed superior angiogenic activity compared to visfatin itself and stimulated mRNA expressions of visfatin and VEGF-A. These results show that the peptides generated by the protein-peptide docking simulation have a more efficient angiogenic activity than the original visfatin.

Data Collection Framework for Context-Aware Virtual Reality Application Development in Unity: Case of Avatar Embodiment.

Virtual Reality (VR) has been adopted as a leading technology for the metaverse, yet most previous VR systems provide one-size-fits-all experiences to users. Context-awareness in VR enables personalized experiences in the metaverse, such as improved embodiment and deeper integration of the real world and virtual worlds. Personalization requires context data from diverse sources. We proposed a reusable and extensible context data collection framework, ManySense VR, which unifies data collection from diverse sources for VR applications. ManySense VR was implemented in Unity based on extensible context data managers collecting data from data sources such as an eye tracker, electroencephalogram, pulse, respiration, galvanic skin response, facial tracker, and Open Weather Map. We used ManySense VR to build a context-aware embodiment VR scene where the user's avatar is synchronized with their bodily actions. The performance evaluation of ManySense VR showed good performance in processor usage, frame rate, and memory footprint. Additionally, we conducted a qualitative formative evaluation by interviewing five developers (two males and three females; mean age: 22) after they used and extended ManySense VR. The participants expressed advantages (e.g., ease-of-use, learnability, familiarity, quickness, and extensibility), disadvantages (e.g., inconvenient/error-prone data query method and lack of diversity in callback methods), future application ideas, and improvement suggestions that indicate potential and can guide future development. In conclusion, ManySense VR is an efficient tool for researchers and developers to easily integrate context data into their Unity-based VR applications for the metaverse.

AEG-1 promotes mesenchymal transition through the activation of Rho GTPases in human glioblastoma cells.

Despite growing evidence indicating that astrocyte elevated gene-1 (AEG-1) plays pivotal roles in tumor progression in various types of human cancers including brain tumors; to date, its role in the regulation of mesenchymal transition is not clear in glioblastoma. In the present study, we investigated the contribution of AEG-1 to stress fiber formation and then the acquisition of mesenchymal characteristics of glioblastoma cells. Gain- and loss-of-function studies in normal immortalized primary human fetal astrocytes (IM-PHFAs) and glioblastoma cells revealed that overexpression of AEG-1 increased expression of mesenchymal markers including N-cadherin and two mesenchymal transition­inducing transcription factors ZEB1 and Slug but decreased epithelial markers E-cadherin and ZO-1. In addition, knockdown of AEG-1 suppressed invasive ability and migration of glioblastoma cells. Overexpression of AEG-1 also induced stress fiber formation and activated the Rho GTPase signaling pathways in glioblastoma cells. Consistently, treatment with an RhoA inhibitor decreased AEG-1-mediated stress fiber formation in glioblastoma cells. Collectively, our findings suggest that AEG-1 promotes mesenchymal transition in glioblastoma through the regulation of the Rho signaling pathway, resulting in tumor invasion, a primary characteristic of malignant brain tumors.

In vitro evaluation of Cordyceps militaris as a potential radioprotective agent.

Radiation is an important component of therapy for a wide range of malignant conditions. However, it triggers DNA damage and cell death in normal cells and results in adverse side-effects. Cordyceps militaris (C. militaris), a traditional medicinal mushroom, produces the bioactive compound, cordycepin (3'-deoxyadenosine) and has multiple pharmacological activities, such as antitumor, antimetastatic, antioxidant and immunomodulatory effects. The present study was undertaken to investigate whether CM-AE, an extract obtained from C. militaris exerts protective effects against radiation-induced DNA damage. The protective effects of CM-AE were compared with those of cordycepin. CM-AE effectively increased free radical scavenging activity and decreased radiation-induced plasmid DNA strand breaks in in vitro assays. CM-AE significantly inhibited the generation of reactive oxygen species (ROS) and cellular DNA damage in 2 Gy irradiated Chinese hamster ovary (CHO)-K1 cells. Moreover, treatment with CM-AE induced similar levels of phosphorylated H2AX in the cells, which reflects the initial DNA double-strand breaks in the irradiated cells compared with the non-irradiated CHO-K1 cells. However, cordycepin did not show free radical scavenging activity and did not protect against radiation-induced plasmid DNA or cellular DNA damage. These results suggest that the free radical scavenging activity of CM-AE contributes towards its DNA radioprotective effects and that the protective effects of CM-AE are much more potent to those of cordycepin. The data presented in this study may provide useful information for the screening of potent radioprotective materials.

Cordycepin-enriched Cordyceps militaris induces immunomodulation and tumor growth delay in mouse-derived breast cancer.

Cordyceps militaris (C. militaris) and its main functional component, cordycepin, has been shown to possess a number of pharmacological activities including immunological stimulation and antitumor effects. However, the pharmacological mechanisms of C. militaris on tumor immunity underlying its antitumor effect have yet to be elucidated. In the present study, we evaluated the antitumor and immunomodulatory effects of C. militaris on FM3A tumor-bearing C3H/He mice, comparing wild-type C. militaris and cordycepin-enriched C. militaris (JLM 0636). The concentration of cordycepin produced by crossbred JLM 0636 was 7.42 mg/g dry weight, which was 7-fold higher than that of wild-type C. militaris. Dietary administration of C. militaris revealed retardation of tumor growth as well as elongation of survival rates of tumor-bearing mice. This effect was more pronounced in JLM 0636. There was a cordycepin-dependent decrease in IL-2 and TGF-ß secretion and an increase in IL-4 secretion without changes in the proliferative responses of concanavalin A-stimulated lymphocytes, which suggested that C. militaris feeding might induce changes in the subpopulations of tumor-derived T lymphocytes. CD4+CD25+ cell population was significantly reduced in the total splenocytes from JLM 0636-administered mice, while CD4+ T cell population remained unchanged. FoxP3+-expressing Treg cells among CD4+CD25+ population showed a similar pattern. On the contrary, CD8+ T cells as well as the IFN-γ expressing CD8+ T cells from tumor-bearing mice were significantly upregulated by the administration of JLM 0636. These results demonstrated the suppressive role of JLM 0636 on the function of Treg cells contributing to tumor specific IFN-γ-expressing CD8+ T cell responses in tumor-bearing mice, which explained the underlying mechanism of the antitumor immunity of cordycepin. Therefore, cordycepin-enriched C. militaris is a promising candidate for an adjuvant in cancer immunotherapy.

Inhibitory effects of Asterina pectinifera extracts on melanin biosynthesis through tyrosinase activity.

The control of melanogenesis is an important strategy in the treatment of abnormal skin pigmentation for cosmetic purposes. The aim of the present study was to investigate the anti-melanogenic effect of Asterina pectinifera (A. pectinifera) extracts by cell-free mushroom tyrosinase assay, cellular tyrosinase assay, melanin content assay and the analysis of related protein expression in melan-a cells. A. pectinifera was extracted with 80% methanol (80-MAP) and further fractionated with hexane (He-AP) and ethyl acetate (EA-AP). In addition, the enzyme extract (En-AP) of A. pectinifera, to which protease was added, was processed. EA-AP and En-AP among A. pectinifera extracts showed strong inhibitory activity against the cell-free mushroom tyrosinase activity. EA-AP and En-AP induced significant inhibition of melanin production and cellular tyrosinase activity. In the action of EA-AP and En-AP on melanogenesis, they reduced the expression of melanogenic genes and proteins including tyrosinase, tyrosinase-related protein-1 (TRP-1) and dopachrome tautomerase (Dct). These results showed that EA-AP and En-AP inhibited melanogenesis by reducing tyrosinase activity and melanin production via subsequent downregulation of tyrosinase-related proteins. The overall results suggest that EA-AP and En-AP among A. pectinifera extracts may be promising candidates for the treatment of hyperpigmentation disorder and useful for self-tanning cosmetic products.

Spray-coated carbon nanotube thin-film transistors with striped transport channels.

We present results for the transfer characteristics of carbon nanotube thin-film transistors (CNT-TFTs) that utilize single-walled carbon nanotube thin-films prepared by direct spray-coating on the substrate. By varying the number of spray-coatings (N(sp)) and the concentration of nanotubes in solution (C(NT)), it was possible to control the conductivity of the spray-coated nanotube thin-film from 129 to 0.1 kΩ/□. Also, by introducing stripes into the channel of the CNT-TFT, and thereby reducing the number of metallic percolation paths between source and drain, it was possible to enhance the on/off current ratio 1000-fold, from 10 to 10(4), demonstrating that it may be possible to utilize spray-coating as a method to fabricate CNT-TFTs for large area switching array applications.

The novel resveratrol analogue HS-1793 induces apoptosis via the mitochondrial pathway in murine breast cancer cells.

Resveratrol (3,4',5 tri-hydroxystilbene), a natural plant polyphenol, has gained interest as a non-toxic chemopreventive agent capable of inducing tumor cell death in a variety of cancer types. Several studies were undertaken to obtain synthetic analogues of resveratrol with potent anticancer activity. The aim of the present study was to investigate the effect of HS-1793 as a new resveratrol analog on apoptosis via the mitochondrial pathway in murine breast cancer cells. A pharmacological dose (1.3-20 µM) of HS-1793 exerted a cytotoxic effect on murine breast cancer cells resulting in apoptosis. HS-1793-mediated cytotoxicity in FM3A cells by several apoptotic events including mitochondrial cytochrome c release, activation of caspase-3 and PARP occurred. In addition, HS-1793 induced collapse of ∆Ψm and enhanced AIF and Endo G release from mitochondria while undergoing apoptosis. These results demonstrate that the cytotoxicity by HS-1793 in FM3A cells can mainly be attributed to apoptosis via a mitochondrial pathway by caspase activation or contributions of AIF and Endo G.

Effect of cordycepin purified from Cordyceps militaris on Th1 and Th2 cytokines in mouse splenocytes.

Cordycepin was purified from a mushroom, Cordyceps militaris, and its effect on Th1 and Th2 cytokines was examined. The level of cytokine induction in mouse splenocytes was estimated after co-inoculation of purified cordycepin and LPS. When 5 microg/ml of purified cordycepin was exposed to mouse splenocytes for 72 h, the level of a Th1 cytokine IL-12 increased by 2.9-fold. The addition of the purified cordycepin to splenocytes also increased the level of Th2 cytokines, IL-4 and IL-10, by 1.9- and 1.8- fold, respectively. Therefore, cordycepin increases the cytokine levels and may contribute to the up-regulation of cellular and humoral immunity.

Fabrication of a flexible and transparent touch sensor using single-walled carbon nanotube thin-films.

Reported herein are the fabrication and demonstration of a flexible and transparent touch sensor using carbon nanotube thin films (CNTFs). The CNTF was fabricated by vacuum filtration and was transferred CNTF to polydimethylsiloxane (PDMS) by water-assisted stamping method. The sheet resistance of the CNTF decreased by approximately 74% after HNO3 treatment. The CNTF touch sensor was fabricated similarly to the conventional four-wire touch screen structures. PDMS was used for the upper plate to absorb the tensile and compressive strain and polyethylene terephthalate (PET) for the lower plate to provide device stability during bending action. The CNTF touch sensor showed high optical transmittance (over 80%) and high sensitivity with the measured touch activation pressure of 23 kPa. Cyclic pressure (38 kPa) was applied at 0.5 Hz and good repeatability was found for several hundred cycles. The results show that the CNTF flexible touch sensor can be applied to future flexible electronic interfaces such as, e-paper and flexible displays.

Effect of hydroxyapatite on bone integration in a rabbit tibial defect model.

BACKGROUND: The aim of the present study was to prepare hydroxyapatite (HA) and then characterize its effect on bone integration in a rabbit tibial defect model. The bone formation with different designs of HA was compared and the bony integration of several graft materials was investigated qualitatively by radiologic and histologic study. METHODS: Ten rabbits were included in this study; two holes were drilled bilaterally across the near cortex and the four holes in each rabbit were divided into four treatment groups (HAP, hydroxyapatite powder; HAC, hydroxyapatite cylinder; HA/TCP, hydroxyapatite/tri-calcium phosphate cylinder, and titanium cylinder). The volume of bone ingrowth and the change of bone mineral density were statistically calculated by computed tomography five times for each treatment group at 0, 2, 4, 6, and 8 weeks after grafting. Histologic analysis was performed at 8 weeks after grafting. RESULTS: The HAP group showed the most pronounced effect on the bone ingrowth surface area, which seen at 4, 6, and 8 weeks after graft (p < 0.05). On comparing the change of bone mineral density the bone ingrowth surface area among the 4 groups, there were no statistically significant differences among the groups found for any period (p > 0.05). On histological examination, the HAP group revealed well-recovered cortical bone, but the bone was irregularly thickened and haphazardly admixed with powder. The HAC group showed similar histological features to those of the HA/TCP group; the cortical surface of the newly developed bone was smooth and the bone matrix on the surface of the cylinder was regularly arranged. CONCLUSIONS: We concluded that both the hydroxyapatite powder and cylinder models investigated in our study may be suitable as a bone substitute in the rabbit tibial defect model, but their characteristic properties are quite different. In contrast to hydroxyapatite powder, which showed better results for the bone ingrowth surface, the hydroxyapatite cylinder showed better results for the sustained morphology.

Functional analysis of six drought-inducible promoters in transgenic rice plants throughout all stages of plant growth.

There are few efficient promoters for use with stress-inducible gene expression in plants, and in particular for monocotyledonous crops. Here, we report the identification of six genes, Rab21, Wsi18, Lea3, Uge1, Dip1, and R1G1B that were induced by drought stress in rice microarray experiments. Gene promoters were linked to the gfp reporter and their activities were analyzed in transgenic rice plants throughout all stages of plant growth, from dry seeds to vegetative tissues to flowers, both before and after drought treatments. In fold induction levels, Rab21 and Wsi18 promoters ranged from 65- and 36-fold in leaves to 1,355- and 492-fold in flowers, respectively, whereas Lea3 and Uge1 were higher in leaves, but lower in roots and flowers, as compared with Rab21 and Wsi18. Dip1 and R1G1B promoters had higher basal levels of activity under normal growth conditions in all tissues, resulting in smaller fold-induction levels than those of the others. In drought treatment time course, activities of Dip1 and R1G1B promoters rapidly increased, peaked at 2 h, and remained constant until 8 h, while that of Lea3 slowly yet steadily increased until 8 h. Interestingly, Rab21 activity increased rapidly and steadily in response to drought stress until expression peaked at 8 h. Thus, we have isolated and characterized six rice promoters that are all distinct in fold induction, tissue specificity, and induction kinetics under drought conditions, providing a variety of drought-inducible promoters for crop biotechnology.

Analysis of five novel putative constitutive gene promoters in transgenic rice plants.

Novel constitutive gene promoters are essential components of crop biotechnology. Our analysis of five such promoters, APX, SCP1, PGD1, R1G1B, and EIF5, in transgenic rice plants is reported here. The five promoter regions were linked to the gfp reporter gene and transformed into rice. Using fluorescent microscopy and q-RT-PCR, promoter activities were analysed in comparison with OsCc1, Act1, and ZmUbi1, previously characterized as strong constitutive promoters. The APX and PGD1 promoters direct high levels of gene expression in all tissues and stages, producing GFP at levels of up to 1.3% of the total soluble protein. PGD1 is particularly active in flowers and mature roots. The R1G1B is active in the whole grain including the embryo, endosperm, and aleurone layer, and thus represents a constitutive promoter with activity in whole seeds that has not been described previously. The ZmUbi1 and R1G1B promoters are markedly less active in young roots and mature leaves whilst the APX, PGD1, OsCc1, and Act1 promoters are highly active in both vegetative and reproductive tissues. Overall, our results demonstrate that APX, PGD1, and R1G1B are novel gene promoters that are highly active at all stages of plant growth with distinct levels of activity.

Protective effect of fermented sea tangle against ethanol and carbon tetrachloride-induced hepatic damage in Sprague-Dawley rats.

Sea tangle has long been used as Korean folk remedy to promote material health, and is one of the popular dietary supplement. This study was designed to evaluate the protective effect of fermented sea tangle (FST) against ethanol and carbon tetrachloride (CCl(4))-induced hepatotoxicity in rats. Sprague-Dawley rats were orally treated with FST (25, 250, 2500 mg/kg/day) with administration of ethanol (5 mL/kg) for 13 weeks and the single intraperitoneal (i.p.) dose of 50% CCl(4) (5 mL/kg/day, CCl(4) in olive oil) at 12 week, and repeated i.p. dose of 20% CCl(4) (2 mL/kg/day) for 1 week. Hepatotoxicity was evaluated by measuring the serum levels of glutamic pyruvate transaminase (GPT), gamma glutamyl transpeptidase (gamma-GT) and malondialdehyde (MDA) as well as the tissue levels of antioxidant enzyme such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Ethanol and CCl(4)-induced the rat liver damage, and significantly increased (p<0.05) the GPT, gamma-GT and MDA levels, and decreased the SOD, CAT and GPx levels. However, treatment with FST could decrease serum GPT, gamma-GT, and MDA levels significantly in plasma, and increase the activities of SOD, CAT, and GPx in liver tissues compared with ethanol and CCl(4)-treated group.

Crystal structure of the periplasmic component of a tripartite macrolide-specific efflux pump.

In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.

Expression and biochemical characterization of the periplasmic domain of bacterial outer membrane porin TdeA.

TolC is an outer membrane porin protein and an essential component of drug efflux and type-I secretion systems in Gram-negative bacteria. TolC comprises a periplasmic alpha- helical barrel domain and a membrane-embedded beta-barrel domain. TdeA, a functional and structural homolog of TolC, is required for toxin and drug export in the pathogenic oral bacterium Actinobacillus actinomycetemcomitans. Here, we report the expression of the periplasmic domain of TdeA as a soluble protein by substitution of the membraneembedded domain with short linkers, which enabled us to purify the protein in the absence of detergent. We confirmed the structural integrity of the TdeA periplasmic domain by size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy, which together showed that the periplasmic domain of the TolC protein family can fold correctly on its own. We further demonstrated that the periplasmic domain of TdeA interacts with peptidoglycans of the bacterial cell wall, which supports the idea that completely folded TolC family proteins traverse the peptidoglycan layer to interact with inner membrane transporters.

Immunostimulating factor isolated from actinobacillus actinomycetemcomitans stimulates monocytes and inflammatory macrophages.

Immunostimulating factor (ISTF) isolated from Actinobacillus actinomycetemcomitans which has been described previously, is distinct from lipopolysaccharide and induces proliferation of B cells. This study was undertaken to investigate whether ISTF might enhance the stimulation of other immune cells. Immunohistochemically, ISTF exhibited a profound stimulating effect on macrophages and dendritic cells as well as B cells in the spleen of BALB/c mice. ISTF was also recognized for its capacity to induce direct activation of mouse macrophages to produce IL-6, TNF-alpha, and NO and MHC class II expression. Therefore, it is postulated that ISTF stimulates macrophages and possibly other cells to produce a wide variety of proinflammatory mediators, which may be involved in the chronicity and tissue destruction of periodontal disease.

Loss of mitochondrial membrane potential and caspase activation enhance apoptosis in irradiated K562 cells treated with herbimycin A.

PURPOSE: We previously reported that herbimycin A (HMA) alters the mode of cell death of K562 cells induced by radiation and enhanced their radiosensitivity. In the present study, we explored the apoptosis-inducing activity of HMA and the fundamental mechanism via which it regulates radiation-induced cell death. MATERIALS AND METHODS: Chronic myelogenous leukemia (CML) cell line K562 was used. For X-irradiation and drug treatment, cells were plated at approximately 2x10(5) cells/ml. Exponentially growing cells were treated with 10 Gy of X-ray using a 6-MeV X-ray machine at a dose rate of 200-300 cGy/min. The cells were treated with 0.25 microM HMA immediately after irradiation and HMA remained for the entire culture period. The modes of cell death were discriminated by morphological changes, analysis of cell cycle, analysis of the mitochondrial events, and the expression of apoptosis-related proteins. RESULTS: Our data demonstrates that radiation induced a significant time-dependent increase of cell death and failed to sustain a prolonged G2 arrest in K562 cells. Radiation-induced cell death caused the accumulation of cyclinB1 and weak nuclear fragmentation, suggesting a mitotic catastrophe. This mitotic catastrophe was dependent upon the mitochondrial permeability transition pore (PTP) opening and was independent of caspase-3. In contrast, K562 cells treated with radiation and HMA had an accelerated cell death and induced a p53-independent apoptosis. This apoptotic pathway was dependent upon an initial hyperpolarization of the mitochondrial inner membrane, following the release of cytochrome c and subsequent caspase-3 activation. CONCLUSIONS: Two mechanisms of radiation-induced cell death in K562 cells, mitotic catastrophe and apoptosis, are regulated through distinct pathways, mitochondria and caspase-independent and -dependent, respectively. The findings of this study may provide new insights into improving the efficiency of radiotherapy in CML patients.

Identification of vasopressin-induced genes in AQP2-transfected MDCK cells by suppression subtractive hybridization.

Arginine vasopressin (AVP) plays a major role in the modulation of water reabsorption in mammalian kidney. In addition to short-term regulation of aquaporin 2 (AQP2) trafficking, AVP also has long-term effects to regulate the expression of AQP2 in renal collecting duct. However, the detailed mechanism of the long-term effects of AVP in kidney remains to be elucidated. We have searched for genes induced by AVP using the polymerase chain reaction-based suppression subtractive hybridization technique in AVP-responsive AQP2-transfected MDCK cells. We found that the expression of the genes such as VIP17/MAL, annexin II, stimulatory GTP binding protein, tubulin, and mitochondrial ATP synthase was induced by AVP treatment for 4h. These results suggest that AVP might induce the expression of several genes related to the apical targeting of newly synthesized AQP2 as well as that of AQP2 for the long-term modification of water permeability in renal collecting duct.

Direct injection of immature dendritic cells into irradiated tumor induces efficient antitumor immunity.

Although there are several ways to load tumor antigens to DCs, in vitro preparation of tumor antigens and manipulation of DCs are usually required. Therefore, to develop a simple antitumor immunization method, we examined if direct injection of DCs into tumor apoptosed by ionizing IR could induce efficient antitumor immunity. Ionizing IR with 15 Gy induced apoptosis in tumor maximally after 6 hr. Injection of DCs i.t. into IR tumor induced strong cytotoxicity of splenocytes against tumor cells compared to i.t. injection of DCs or ionizing IR of tumor, both of which induced weak cytotoxicity. In an animal study, i.t. injection of DCs into IR tumor induced therapeutic antitumor immunity against a tumor established at a distant site. Moreover, when TNF-alpha or LPS was added as a danger/maturation signal to DC suspension before i.t. injection, antitumor immunity was significantly potentiated compared to a group treated with i.t. injection of DCs into IR tumor. Our results suggest that injection of DCs into tumor apoptosed by ionizing IR might be a simple and efficient method of immunization against tumor.