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Aging is considered to be a functional retardation of continuous xenobiotic responses over a lifetime after the developmental period; thus, the effects of ionizing radiation over a lifetime may be somewhat accounted for by a modifier of aging effects. This study was conducted to evaluate the possible/synergic effects of radiation during aging by determining cell-cycle parameters of hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs), such as the percent of cells in cycling, the generation doubling time, and the cumulative cycling-cell fraction, by bromodeoxyuridine-ultraviolet assay, which enables the determination of their cycling capacity in vivo. Colony-forming progenitor cells, such as colony-forming unit (CFU)-granulocyte/macrophage (GM), CFU in the spleen on day 9 (CFU-S9), and CFU-S on day 13 (CFU-S13) for mature, less mature, and immature HPCs, respectively, were evaluated in young and old mice (6 weeks and 21 months of age, respectively) with or without 2-Gy whole-body irradiation and a 4-week recovery period. Then, cell-cycle parameters were evaluated and compared. As a result, the generation doubling time of all types of HPC was prolonged by the irradiation in both young and old mouse groups, except that of CFU-S13 in old mice, which showed acceleration of the cell cycle following the irradiation. In addition, only CFU-S13 in irradiated old mice showed a significant increase in the cumulative cycling-cell-fraction ratio. Significant changes due to the effects of aging and irradiation on HPCs were observed only in the immature HPCs, i.e., the cell cycle of immature HPCs was suppressed by aging without irradiation and was, in contrast, accelerated as the cells recovered from radiation-induced damage. This suggests that the mechanisms of peripheral blood recovery after 2-Gy whole-body irradiation are markedly different between young and old mice, although 21-month-old mice showed almost the same level of recovery as the young mice.

Hideki Hirakata, Kosaku Nitta, Masaaki Inaba, Tetsuo Shoji, Hideki Fujii, Shuzo Kobayashi, Kaoru Tabei, Nobuhiko Joki, Hiroki Hase, Masato Nishimura, Shigeyuki Ozaki, Yuji Ikari, Yoshitaka Kumada, Kazuhiko Tsuruya, Shouichi Fujimoto, Tohru Inoue, Hiroyoshi Yokoi, Sumio Hirata, Kazuaki Shimamoto, Kiyotaka Kugiyama, Takashi Akiba, Kunitoshi Iseki, Yoshiharu Tsubakihara, Tadashi Tomo, and Tadao Akizawa

Surface chemical properties of a phospholipid, dilauroylphosphatidylcholine (DLPC), in two ionic liquids (ILs), 1-butyl-3-methylimidazolium tetrafluoroborate (bmimBF(4)) and hexafluorophosphate (bmimPF(6)), were investigated by means of surface tension, dynamic light-scattering, and freeze-fracture transmission electron microscopy. It was found that DLPC shows finite solubility in the ILs and spontaneously forms vesicles with size distribution around 400 nm in diameter above the critical vesicular concentration (CVC) of 0.040 wt.% (in bmimBF(4)) and approx. 0.08 wt.% (in bmimPF(6)). Other than the CVC value, anion specificity of the ILs was also seen in the temperature effect on the vesicular aggregation; that is, a temperature-induced reversible aggregation was observed in bmimPF(6), but not in bmimBF(4). The differences in the vesicular stability against the temperature-induced aggregation could be attributed to differences in the interaction between anion species of the ILs and zwitterionic phosphatidylcholine head groups. The apparent molecular area occupied by DLPC at the air/solution interface was estimated to be 0.37 nm(2) in bmimBF(4) and 0.20 nm(2) in bmimPF(6) by applying the Gibbs adsorption equation. These values are much smaller than the molecular area of 0.69 nm(2) reported for the hydrated DLPC bilayer of lamellar liquid-crystalline phase. This result is not consistent with the traditional Gibbs adsorption model, but can be interpreted in terms of a picture for the surface adsorption of soluble amphiphiles proposed by Moroi et al. Differential scanning calorimetric study is also reported regarding the phase transition behavior of DLPC bilayer solvated by the ILs.

Surface adsorption and aggregation behavior of polyoxyethylene (POE)-type nonionic surfactants in a room temperature ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate (bmimPF6), were investigated by means of surface tension, 1H NMR, and dynamic light-scattering (DLS) measurements. The maximum surface excess in bmimPF6 is much lesser than those observed for aqueous solution, reflecting a much weaker solvophobicity exhibited by the surfactant hydrocarbon chain in the ionic liquid compared with the hydrophobicity experienced in water. Compared with another ionic liquid with different anion species, 1-butyl-3-methylimidazolium terafluoroborate (bmimBF4), the surfactant solution in bmimPF6 brings about higher critical micelle concentration (cmc), smaller micellar aggregation number, and weaker dependence of cmc on the surfactant hydrocarbon chain length. This indicates that the solvophobic interaction between surfactant hydrocarbon chains acts more weakly in bmimPF6 than in bmimBF4. The weaker solvophobic effect in bmimPF6 is also appreciable in the thermodynamic parameters for micelle formation derived from temperature dependence of cmc, and would be attributed to weaker attractive interaction between bmim cation and PF 6 - ion compared with that between bmim cation and BF 4 - ion. Present results give an insight into a better understanding of the importance of anion species for the properties of ionic liquids as a solvent to support a self-assembly of amphiphilic molecules.

症例は68歳，男性．不消化性の下痢，曖気の精査加療目的で入院となった．腹部CTで下行結腸～脾彎曲にかけて13cm大の腫瘤性病変を認めた．下部消化管内視鏡検査では下行結腸に隆起を主体とする5型病変を認め，ファイバーは潰瘍底より容易に胃内へと挿入された．腫瘍からの生検で高分化型腺癌と診断された．胃結腸瘻を形成した進行下行結腸癌と診断し，左半結腸切除術，D3リンパ節郭清，胃全摘，膵尾部・脾臓合併切除術を施行した．最終診断はmuc，si，n0，ly0，v0，ow(－)，aw(－)，stageIIで，手術根治度Aであった．われわれは内視鏡が通過することで診断できた胃結腸瘻を形成した下行結腸癌の1例を経験したので，自験例を加えた本邦報告25例の集計および文献的考察を行った．

Phase behaviors of the binary mixtures composed of ethylene carbonate (EC) and aliphatic alcohols, ω-phenyl alcohols, and alkylbenzenes were investigated. In addition, heat of solution of EC into these organic solvents was measured. The EC/methanol and EC/ethanol systems gave homogeneous solution at the temperature above their liquidus lines, while the mixtures of EC and alcohols with longer alkyl chain showed a miscibility gap in a liquid phase and provided the monotectic-type phase diagram. The liquid–liquid phase separation region expanded with the increase in the alkyl chain length. A similar phase behavior was also observed for the mixtures of EC and alkylbenzenes. On the other hand, the EC mixtures with ω-phenyl alcohols showed no miscibility gap in a liquid phase at least up to 4-phenylbutan-1-ol which has C4 alkyl chain intervening between phenyl and hydroxyl groups. This result demonstrates that both of the hydroxyl and phenyl groups act to facilitate the mixing of aliphatic compounds with EC. The phase behavior of these EC mixtures was analyzed applying the modified regular solution model in which the pair interaction energy was regarded as free energy. The model calculation with the use of heat of solution of EC at infinite dilution as the pair interaction enthalpy reproduced well both of the experimentally obtained liquidus line and mutual solubility curve as well as monotectic point.

症例は62歳，男性．37年前に十二指腸潰瘍に対して胃切除術の既往がある．1カ月前から嘔気・嘔吐を認めるようになり近医受診した．腹部CTで吻合部に腫瘤影と輸入脚の著明な拡張を認めた．同日の上部消化管内視鏡検査では吻合部に全周性の3型腫瘍を認め，残胃癌による輸入脚閉塞症と診断し緊急手術を施行した．前回手術はBillroth-II法の結腸後経路で再建されており，Treitz靱帯から吻合部の空腸は暗赤色を呈し著明に拡張していた．残胃全摘術，膵尾部・脾臓合併切除術を施行した．治癒切除であったが，術後3カ月で胃癌死された．残胃癌による輸入脚閉塞症の本邦論文報告7例について，集計および文献的考察を行った．

In this study, Trp53-deficient and wild-type mice of both C57BL/6 and C3H/He strains were exposed to benzene (33, 100, and 300 ppm; 6h/day, 5 days/week for 26 weeks) and then observed for lifetime. As results, first, the incidence of nonthymic lymphomas in C57BL/6 mice and acute myeloid leukemias (AMLs) in C3H/He mice showed linear responses at the lower exposure level in Trp53-deficient mice; second, the incidence of thymic lymphomas in C57BL/6 mice and nonthymic lymphomas in C3H/He mice increased without a plateau-like ceiling; thus, the former equivocal induction of hematopoietic neoplasms (HPNs) in the case of low-dose benzene exposure was assumed to be based on the DNA repair potential in wild-type mice, and the latter limited increase in HPNs in the case of high-dose benzene exposure was considered to be due to excessive apoptosis in wild-type mice. Concerning the incidence of AMLs, though a dose of 300 ppm benzene inhalation induced 9% AMLs in wild-type C3H/He mice-AML-prone, it induced AMLs in 38% of Trp53-deficient C3H/He mice. Because AMLs were also observed in Trp53-deficient mice, including in the C57BL/6 mice, benzene exposure may also be a potent inducer of AMLs in mice with some strain differences. In the present study, to elucidate the hematopoietic stem cell-specific, aryl hydrocarbon-receptor-related low-dose adverse effect, global gene expression in the bone marrow was analyzed at 28 days after 2-week-intermittent exposure to 150 mg/kg b.w. benzene, by gavage, i.e., equivalent to the above inhalation protocol with 300 ppm. We observed two conceptually different gene expression profiles; "common gene profiles" (CGPs) shared among mice in each group, and "stochastic gene profiles" (SGPs), i.e., unique union genes from one individual mouse to another. The CGPs of the experimental group and the SGPs of each individual mouse were separately characterized by individual assay. Concerning the CGPs, reciprocal strain differences between C3H/He and C57BL/6 mice in expression gene profiles, both plausible for leukemogenesis, were identified; namely, dominant downmodulations of Sltm and Cryl1, related to suppression of apoptosis and genomic instability in C3H/He mice, respectively, and dominant downmodulations of Atrx/rad54 and Kdm2a, related to a decrease in DNA repair and genomic instability, respectively, in C57BL/6 mice. These findings imply that these reciprocal gene expression differences induced by benzene exposure may lead each strain to undergo different hematopoietic neoplastic pathways. In contrast, each individual mouse often shows a unique SGP. SGPs often include transcription factors, which regulate reciprocal signaling pathways including further SGPs. Among them, apoptosis-related genes expressed in C57BL/6 mice and those in C3H/He mice were attributable to different combinations of SGPs. Such stochastic case-by-case gene expression may be in good agreement with the individual and strain differences observed following benzene exposure. Because gene chip microarray techniques can elucidate stochastic changes in gene expression profiles, possible stochastic toxicology and its future role are discussed.

The melting behavior of polyethyleneglycol dodecyl ethers (C 12 E 6 , C 12 E 7 , and C 12 E 8 ) in a room temperature ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate (bmimBF 4 ), was investigated by means of differential scanning calorimetry (DSC). The melting temperature as a function of the surfactant concentration, combined with the cmc curve and cloud point curve, provided phase diagrams for the surfactant/bmimBF 4 mixtures in solvent-rich region. The characteristic feature of the mixtures is an existence of the Krafft temperature which is usually not observed with aqueous solutions of nonionic surfactants. The heat of fusion as a function of the surfactant concentration provided the interaction energy between the surfactant and bmimBF 4 . The interaction energy shows a linear dependence on the length of polyoxyethylene (POE) chain of the surfactants, which suggests that the solvation takes place around the POE chain.

This research focused on three major questions regarding benzene-induced hematopoietic neoplasms (HPNs). First, why are HPNs induced equivocally and at only threshold level with low-dose benzene exposure despite the significant genotoxicity of benzene even at low doses both in experiments and in epidemiology? Second, why is there no linear increase in incidence at high-dose exposure despite a lower acute toxicity (LD50 > 1000 mg/kg body weight; WHO, 2003, Benzene in drinking-water. Background document for development of WHO Guidelines for Drinking-Water Quality)? Third, why are particular acute myeloid leukemias (AMLs) not commonly observed in mice, although AMLs are frequently observed in human cases of occupational exposure to benzene? In this study, we hypothesized that the threshold-like equivocal induction of HPNs at low-dose benzene exposure is based on DNA repair potential in wild-type mice and that the limited increase in HPNs at a high-dose exposure is due to excessive apoptosis in wild-type mice. To determine whether Trp53 deficiency satisfies the above hypotheses by eliminating or reducing DNA repair and by allowing cells to escape apoptosis, we evaluated the incidence of benzene-induced HPNs in Trp53-deficient C57BL/6 mice with specific regard to AMLs. We also used C3H/He mice, AML prone, with Trp53 deficiency to explore whether a higher incidence of AMLs on benzene exposure might explain the above human-murine differences. As a result, heterozygous Trp53-deficient mice of both strains showed a nonthreshold response of the incidence of HPNs at the lower dose, whereas both strains showed an increasing HPN incidence up to 100% with increasing benzene exposure dose, including AMLs, that developed 38% of heterozygous Trp53-deficient C3H/He mice compared to only 9% of wild-type mice exposed to the high dose. The detection of AMLs in heterozygous Trp53-deficient mice, even in the C57BL/6 strain, implies that benzene may be a potent inducer of AMLs also in mice with some strain differences.

The cloud point phenomenon has been investigated for the solutions of polyoxyethylene (POE)-type nonionic surfactants (C(12)E(5), C(12)E(6), C(12)E(7), C(10)E(6), and C(14)E(6)) in 1-butyl-3-methylimidazolium tetrafluoroborate (bmimBF(4)), a typical room temperature ionic liquid (RTIL). The cloud point, T(c), increases with the elongation of the POE chain, while decreases with the increase in the hydrocarbon chain length. This demonstrates that the solvophilicity/solvophobicity of the surfactants in RTIL comes from POE chain/hydrocarbon chain. When compared with an aqueous system, the chain length dependence of T(c) is larger for the RTIL system regarding both POE and hydrocarbon chains; in particular, hydrocarbon chain length affects T(c) much more strongly in the RTIL system than in equivalent aqueous systems. In a similar fashion to the much-studied aqueous systems, the micellar growth is also observed in this RTIL solvent as the temperature approaches T(c). The cloud point curves have been analyzed using a Flory-Huggins-type model based on phase separation in polymer solutions.

Previously, we found an aryl hydrocarbon receptor (AhR)-transmitted benzene-induced hematotoxicity; that is, AhR-knockout (KO) mice did not show any hematotoxicity after benzene exposure [Yoon, B.I., Hirabayashi, Y., Kawasaki, Y., Kodama, Y., Kaneko, T., Kanno, J., Kim, D.Y., Fujii-Kuriyama, Y., Inoue, T., 2002. Aryl hydrocarbon receptor mediates benzene-induced hematotoxicity. Toxicol. Sci. 70, 150-156]. Furthermore, our preliminary study showed a significant attenuation of benzene-induced hematopoietic toxicity by AhR expression, when the bone marrow (BM) of mice was repopulated with AhR-KO BM cells [Hirabayashi, Y., Yoon, B.I., Li, G., Fujii-Kuriyama, Y., Kaneko, T., Kanno, J., Inoue, T., 2005a. Benzene-induced hematopoietic toxicity transmitted by AhR in the wild-type mouse was negated by repopulation of AhR deficient bone marrow cells. Organohalogen Comp. 67, 2280-2283]. In this study, benzene-induced hematotoxicity and its nullification by AhR-KO BM cells were further precisely reevaluated including the duration of the effect after benzene treatment and recovery after the cessation of exposure. Exposure routes, namely, intraperitoneal (i.p.) injection used in our previous study and intragastric (i.g.) administration used in this study, were also compared in terms of their toxicologic outcomes. From the results of this study, mice that had been lethally irradiated and repopulated with BM cells from AhR-KO mice essentially did not show any benzene-induced hematotoxicity. The AhR-KO BM cells nullified benzene-induced toxicities in notably different hematopoietic endpoints between the i.p. treatment and the i.g. treatment; however, the number of granulo-macrophage colony-forming unit in vitro (CFU-GM) was a common target parameter, the benzene-induced toxicity of which was nullified by the AhR-KO BM cells.

Irradiation of mice at doses of 1-1.5 Gy induced a predominant regeneration of the B-cell lineage but suppressed the regeneration of the myeloid lineage. The mechanisms underlying such reciprocal regulation of regeneration and the relationship between the two lineages remain unclear. Because the predominant regeneration of the B-cell lineage observed is considered to depend on the stromal cell function, and because the impairment of such stromal function may nullify such reciprocal responses, mouse models of senescent stromal cell impairment (SCI) and the less senescent stage of SCI (non-SCI) were compared to elucidate the mechanisms underlying the reciprocal regulation of both lineages after radiation exposure. In non-SCI mice irradiated with 1 Gy, the numbers of B-lymphocyte progenitor (CFU-preB) and granulocyte-macrophage progenitor (CFU-GM) cells in the bone marrow decreased rapidly during the first 24 h. Then the number of CFU-preB cells in the bone marrow promptly recovered from the nadir and exceeded the pretreatment level, whereas that of CFU-GM cells remained lower than the pretreatment level. The expression of genes encoding positive regulators of the B-lymphoid lineage [interleukin (IL)10, Flt3 ligand and IL7] was up-regulated; in contrast, expression of the positive regulators of the myeloid lineage [granulocyte macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF)] was down-regulated. In SCI mice irradiated with 1 Gy, the oscillatory changes in the numbers of femoral CFU-preB and CFU-GM cells and in the expression levels of cytokine genes were less marked than those in the non-SCI mice. These results thus imply that the reciprocal regeneration depends on the up-regulation of IL10, Flt3 ligand and IL7 expression and the down-regulation of GM-CSF and SCF expression in the bone marrow, possibly depending on the hematopoietic microenvironment.

Aqueous solutions of long-chain imidazolium ionic liquids have been investigated by surface tension and steady-state fluorescence measurements at room temperature (298 K). The micelle aggregation number (Nagg) was obtained by pyrene fluorescence quenching method. From the surface tension data, critical micelle concentration (cmc), surface tension at the cmc (γcmc), adsorption efficiency (pC20), and effectiveness of surface tension reduction (Πcmc), were determined. Moreover, applying the Gibbs adsorption isotherm, maximum surface excess concentration (Γmax) and minimum surface area per molecule (Amin) at the air–water interface were estimated. The effect of sodium halides, NaCl, NaBr, and NaI, on the surface activity was also investigated. The addition of salts decreases significantly both cmc and γcmc, and the dependence of the salt effect on the anion species is analogous for the case of conventional ionic surfactants. Due to the bulkiness of the imidazolium head group, the microenvironment in long-chain imidazolium ionic liquid micelles exhibits higher polarity compared with the corresponding CnTAB micelles, and the micelle aggregation numbers (Nagg) are smaller than those of CnTAB.

The study examined the feasibility of screening for hepatotoxicity by an in vitro gene expression analysis using rat primary hepatocytes and Affymetrix Rat Toxicology U34 arrays. Hepatocytes were exposed for 6 or 24 h to eight drugs, with different mechanisms of hepatotoxicity, at one third of the cytotoxic concentration TC50, i.e. acetaminophen, cyclophosphamide, clofibrate, chlorpromazine, lithocholic acid, cisplatin, diclofenac and disulfiram. The types of transcriptional changes observed in this study were generally consistent with previously reported in vivo data, although there were some differences. In hierarchical cluster analysis, drugs formed clusters depending on their mode of toxicity against cells. The number of transcripts affected by the cholestatic hepatotoxicants (lithocholic acid and chlorpromazine) or the drugs that rarely cause of hepatotoxicity (cisplatin, diclofenac and disulfiram) were limited compared with the other drugs (acetaminophen, clobifibrate and cyclophosphamide), where they did not induce transcriptional changes apparently related to toxicity. It is concluded that in vitro gene expression analysis of hepatocytes using microarray is a useful tool for evaluating the toxicological profile of drugs and in screening for the direct toxicity of drugs against hepatocytes.

Purification of perfluoro-n-alkanoic acids (C(n)F(2n+1)COOH, n=7, 9, 11, 13, 15 and 17) was made by repeated recrystallizations from n-hexane/acetone mixed solvent, and their purity was found to be more than 99.5% by GC-MS, NMR, and elemental analysis. The thermal behaviors such as melting point and enthalpy change of fusion were investigated using differential scanning calorimetry (DSC). The melting point monotonously increased with increasing carbon number (n) of the acids, while the enthalpy change showed irregularity at n=14. The crystal structure of these acids was found to be dependent upon solvent used for recrystallization; that is, the acids recrystallized from the above solvent becomes more stable energetically, indicating their higher enthalpy change of fusion than that of the solidified acids from fused ones. The solid state was also found to vary depending upon the thermal history, indicating that a few crystal structures of the solid state are quite similar energetically. The melting points (T(m)) of perfluoro-n-alkanoic acids are higher than those of corresponding n-alkanoic acids, and the difference in T(m) increases with increasing carbon number in the acids.

Since malignant tumors are life-threatening, the death rate from these diseases is high, and existing therapies have limited effectiveness, it is desired to provide new effective anticancer drugs to tumor patients sooner. However, there is no guideline regarding non-clinical safety studies on the development of anticancer drugs required for the first in human clinical trials and for the approval applications in Japan. Then, the Ministry of Health, Labour and Welfare (MHLW) established the collaboration group including regulatory, academic and industrial scientists to prepare the guideline on the non-clinical safety evaluation of anticancer drugs in 2004. As a guide for basic concept of non-clinical safety studies on anticancer drugs, the "Points to Consider" document was prepared by this group in 2007.

Electrical conductivity was measured for aqueous solutions of long-chain imidazolium ionic liquids (IL), 1-alkyl-3-methylimidazolium bromides with C(12)-C(16) alkyl chains. The break points appeared in specific conductivity (kappa) vs concentration (c) plot indicates that the molecular aggregates, i.e., micelles, are formed in aqueous solutions of these IL species. The critical micelle concentration (cmc) determined from the kappa vs c plot is somewhat lower than those for typical cationic surfactants, alkyltrimethylammonium bromides with the same hydrocarbon chain length. The electrical conductivity data were analyzed according to the mixed electrolyte model of micellar solution, and the aggregation number, n, and the degree of counter ion binding, beta, were estimated. The n values of the present ILs are somewhat smaller than those reported for alkyltrimethylammonium bromides, which may be attributed to bulkiness of the cationic head group of the IL species. The thermodynamic parameters for micelle formation of the present ILs were estimated using the values of cmc and beta as a function of temperature. The contribution of entropy term to the micelle formation is superior to that of enthalpy term below about 30 degrees C, and it becomes opposite at higher temperature. This coincides with the picture drawn for the micelle formation of conventional ionic surfactants.

The surface behaviors of 3-nonylphenoxy-2-hydroxyl-propyl-trimethyl-ammonium bromide (RTAB), a cationic surfactant, are examined by dynamic surface tensions (DSTs), π–A isotherms and molecular dynamics simulations as a function of pH. While the DST remains almost constant when the pH of the solutions changes from 7.0 to 3.0, which decreases remarkably in alkaline solutions. The results of π–A isotherms indicate that RTAB molecule has a much larger cross-sectional area at the air–water interface in alkaline solutions than that in acidic or neutral solutions. Molecular dynamics (MD) simulation studies revealed that the hydrocarbon chains of the surfactant molecules adsorbed at the air–water interface become conformationally more complex under alkaline solutions than in neutral or acidic conditions. These abnormal surface behaviors of RTAB in alkaline solutions are explained by assuming the binding of a hydroxide ion to the hydroxyl group in RTAB molecule.

Mice exposed to a lethal dose of radiation were repopulated with heterozygous p53(+/-) (TRP53(+/-)) bone marrow cells and then exposed to doses of 1, 3 and 5 Gy 1 month later. This resulted in the transplanted bone marrow-specific diseases other than competitively induced nonhematopoietic neoplasms. Interestingly, the present study showed a high frequency of stem cell leukemia, i.e., leukemias characterized by a lack of differentiation due also to p53 deficiency, even after 5 Gy irradiation. The frequencies of stem cell leukemias (and those of total hematopoietic malignancies) were 16% (24%) at 1 Gy and 45% (75%) at 3 Gy. Furthermore, markedly high incidences of stem cell leukemias were observed at 5 Gy in p53(+/-) mice, i.e., 87% (100%) in the transplantation assay and 60% (83.3%) in the whole-body assay, whereas a conventional whole-body assay induced only 14% in wild-type mice. The high incidence of stem cell leukemias observed in this study using heterozygous p53-deficient mice agrees with results of a previous study of homozygous p53-deficient mice and is consistent with the high frequency of loss of heterozygosity in the p53 wild-type allele observed in leukemias. This suggests that the target cells for radiation-induced stem cell leukemias may be p53-deficient hematopoietic stem cells.

The National Science Museum (NSM) in Japan has recently completed a project using different types of visitor-oriented digital technologies. With sponsorship from the Japan Society for the Promotion of Science (JSPS), the NSM team carried out a four-year study to examine how digital technologies can be used to enhance as well as educationally enrich a visit to a science and natural history museum. Two types of devices, a handheld audio guide and an integrated circuit card, were tested in the museum. The results of the project are described along with suggestions for designers and educators who are seeking to add similar devices to their museums.

Phase behavior of aqueous mixture of imidazolium ionic liquid, 1-dodecyl-3-methylimidazolium bromide, was investigated by means of differential scanning calorimetry and polarized optical microscopy. The mixture forms two types of lyotropic liquid-crystalline gels, one is composed of lamellar phase and the other is of hexagonal phase. T – X phase diagram of the mixture was constructed, which defines the regions of various phases appearing in this mixture.

Objective Reactive oxygen species (ROS), generated following benzene exposure, are considered to trigger the development of hematopoietic neoplasms, although little supporting evidence has been found. In this study, we examined whether the experimental elimination of ROS generated following benzene exposure prevents the development of benzene-induced hematopoietic disorders to clarify the mechanism underlying the development of benzene-induced hematopoietic disorders. Methods C57BL/6 mice, overexpressing human thioredoxin (h-Trx-Tg), were used to examine the possible nullification of ROS induction following benzene exposure. The experimental group was exposed to 300 ppm benzene 6 hours/day, 5 days/week, for 26 weeks, and lifetime observation followed by molecular and histopathological examinations were carried out. Results The present study using h-Trx-Tg mice showed a complete suppression of the development of thymic lymphoma induced by benzene inhalation (0% in h-Trx-Tg vs 30% in wild-type (Wt) mice). This was associated with a 48% decrease in the incidence of clastogenic micronucleated reticulocyte induction in the h-Trx-Tg mice compared with the Wt control after 2 weeks of inhalation. As underlying mechanisms, the attenuation of oxidative stress was accompanied by a complete abrogation of hemato-lymphoid toxicity, as shown by the upregulation of the activity of superoxide-dismutase, and a consequently stable ROS level, as determined by cell sorting using 2′, 7′-dichlorodihydrofluorescein diacetate, along with a significant attenuation of the overexpression of a cell cycle–dependent kinase inhibitor, p21. Conclusion The attenuation of benzene-induced oxidative stress and that of the consequent lymphomagenesis were observed for the first time, and these indicate a role of oxidative stress in benzene-induced clastogenesis and lymphomagenesis. (These attenuations were not seen in nonthymic lymphomas, and no leukemias developed in C57BL/6 used in this study.) During the constitutive overexpression of h- Trx , the expression of aryl-hydrocarbon receptor in h-Trx-Tg mice was downregulated, which may also contribute to the attenuation.

Previous fate mapping analysis, using Cre recombinase driven by the Mesp1 locus, revealed that Mesp1 is expressed in almost all of the precursors of the cardiovascular system, including the endothelium, endocardium, myocardium, and epicardium. Mesp1-nonexpressing cells were found to be restricted to the outflow tract cushion and along the interventricular septum (IVS), which is a location that is suggestive of specialized cardiac conduction system (CCS). In our current study, we examined the identity of these IVS cells by using the pattern of beta-galactosidase activity in CCS-lacZ mice. In addition, by crossing Mesp1-Cre and floxed GFP reporter mice with CCS-lacZ mice, we have calculated that approximately 20% of the ventricular CCS within the IVS corresponds to Mesp1-nonexpressing cells. These data suggest that the ventricular CCS is of heterocellular origin. Furthermore, we indicate a possibility that a population of the cells that contribute to the ventricular CCS might be distinguished at an early stage of development.

This is the report of the first workshop "Validation of Toxicogenomics-Based Test Systems" held 11-12 December 2003 in Ispra, Italy. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and organized jointly by ECVAM, the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). The primary aim of the workshop was for participants to discuss and define principles applicable to the validation of toxicogenomics platforms as well as validation of specific toxicologic test methods that incorporate toxicogenomics technologies. The workshop was viewed as an opportunity for initiating a dialogue between technologic experts, regulators, and the principal validation bodies and for identifying those factors to which the validation process would be applicable. It was felt that to do so now, as the technology is evolving and associated challenges are identified, would be a basis for the future validation of the technology when it reaches the appropriate stage. Because of the complexity of the issue, different aspects of the validation of toxicogenomics-based test methods were covered. The three focus areas include a) biologic validation of toxicogenomics-based test methods for regulatory decision making, b) technical and bioinformatics aspects related to validation, and c) validation issues as they relate to regulatory acceptance and use of toxicogenomics-based test methods. In this report we summarize the discussions and describe in detail the recommendations for future direction and priorities.

Hematopoiesis in the bone marrow (BM) and spleen is controlled by stromal cells. Inflammation promotes myelopoiesis and simultaneously suppresses B lymphopoiesis. However, the role of the reciprocal regulation of myelopoiesis and B lymphopoiesis by stromal cells during inflammation is not fully understood. We investigated inflammation-induced alteration of hematopoietic regulation in lipopolysaccharide (LPS)-treated mice. C57BL/6 female mice were intravenously injected with a single, 5-µg dose of LPS, which induced a rapid decrease in the number of granulocyte-macrophage progenitors (colony-forming unit granulocyte-macrophage; CFU-GM) and B cell progenitors (CFU-preB) in BM. The CFU-GM count rapidly recovered, whereas the recovery of CFU-preB was delayed. LPS induced a marked increase in the number of CFU-GM but not in the number of CFU-preB in spleen. After LPS treatment, gene expression levels of positive regulators of myelopoiesis such as granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in BM and spleen were markedly upregulated whereas levels of positive regulators for B lymphopoiesis such as stromal cell-derived factor (SDF)-1, stem cell factor (SCF), and IL-7 remained unchanged. Meanwhile, the negative regulator of B lymphopoiesis tumor necrosis factor (TNF)-α was markedly up-regulated. The number of CFU-GM in S-phase in BM increased after LPS treatment, whereas the number of CFU-preB in S-phase decreased. These results suggest that LPS-activated stromal cells induce positive-dominant regulation of myelopoiesis and negative-dominant regulation of B lymphopoiesis, which facilitates emergency myelopoiesis during inflammation by suppressing B lymphopoiesis, thereby contributing to the host defense against infection.

The effect of sucrose on the structure of molecular assemblies formed in an MO/H2O mixture has been studied using a small-angle X-ray scattering method. It was found that the phase transition Ia3d → Pn3m → HII occurs with increased sucrose concentration in the mixture with the composition 70 wt% MO at 20 °C. This structural change induced by sucrose addition would be ascribed to a cosmotropic property of sucrose, which leads to the dehydration of the MO head group and hence causes a decrease in the effective area occupied by the MO molecule at the polar/apolar interface, which facilitates the formation of molecular assemblies with a high curvature for the reversed liquid-crystal mesophase.

Micelle formation in aqueous solution of novel hybrid surfactants with phosphate head group was investigated. The critical micelle concentrations (CMC) were determined as a function of temperature by surface tension measurements. The results of electrical conductivity measurements were analyzed according to the mixed electrolyte model of the micellar solution to estimate the micellar aggregation number and the degree of counter-ion binding on the micelle. Thermodynamic parameters for the micelle formation were estimated on the basis of these results. Discussion is given on the micelle formation of the present surfactant species comparing with the sulfonate-type hybrid surfactants.

The present study was performed to assess the protective role of thioredoxin/adult T-cell leukemia-derived factor (TRX/ADF) on the liver and brain cell damages induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in ADF wild-type (WT) and transgenic (Tg) mice. The ADF WT and Tg mice were intraperitoneally injected with a single dose of TCDD (150 μg/kg body weight). One day after the treatment, the liver and brain tissues were examined electron microscopically to evaluate the cellular toxicity. In the ADF WT mice, marked reduction of subcellular components, such as mitochondria, rough endoplasmic reticula, and glycogen granules, as well as swelling of the remaining mitochondria, were evident in the liver cells. However, attenuation of these changes was evident in TCDD-treated TRX/ADF mice. Similar subcellular changes noted in the neuronal cells of TCDD-treated WT mice were also attenuated in Tg mice. The results suggest that oxidative cellular damage contributes to the acute toxicity induced by TCDD and that TRX/ADF protects against it.

We have previously demonstrated that in mice, the decrease in serum thyroxine (T(4)) level by polychlorinated biphenyls (PCBs) occurs without an increase in the UDP-glucuronosyltransferase (T(4)-UDP-GT) for T(4) glucuronidation, although the PCB-induced decrease in rats is generally thought to occur through induction of T(4)-UDP-GT, UGT1A1, and UGT1A6. In the present study, to further clarify the relationship between the decrease in serum T(4) level and the increase in UGT1A activity by PCB in rats, we examined the relationship using Wistar rats and Gunn rats, a mutant strain of Wistar rats deficient in UGT1A isoforms. The serum total T(4) level was markedly decreased not only in the Wistar rats but also in the Gunn rats 4 days after treatment with a PCB, Kanechlor-500 (KC500, 100 mg/kg) or 2,2',4,5,5'-pentachlorobiphenyl (PentaCB, 112 mg/kg), and there was no significant difference in magnitude of the decrease between the two rat strains. At the same time, the level and activity of T(4)-UDP-GT were significantly increased by treatment with either KC500 or PentaCB in Wistar rats but not in Gunn rats. In addition, no significant change in the level of serum total triiodothyronine (T(3)) and thyroid-stimulating hormone by the KC500 treatment was observed in either Wistar or Gunn rats. Furthermore, significant decrease in the activity of hepatic type-I deiodinase, which mediates the deiodization of T(4) and T(3), by treatment with KC500 or PentaCB was observed in both Wistar and Gunn rats. From the serum of KC500- or PentaCB-treated Wistar and Gunn rats, mono- and di-hydroxylated PCB metabolites, which would bind to T(4) binding serum protein (transthyretin), were detected. In conclusion, the present results suggest that the decrease in serum total T(4) level by either KC500 or PentaCB in Gunn rats was not dependent on the increase in hepatic T(4)-UDP-GT activity. The findings further suggest that the PCB-mediated decrease in serum T(4) level might occur, at least in part, through formation of the hydroxylated PCB metabolites. Furthermore, even in Wistar rats, the PCB-mediated decrease in serum T(4) level might occur not only through the increase in hepatic T(4)-UDP-GT but also via formation of hydroxylated PCB metabolites.

The aqueous phase behavior of heptaethylene glycol dodecyl ether (C12E7) was investigated in the presence of sodium salts of Cl−, I−, and ClO4−. Pseudo binary T–X phase diagrams were constructed for these mixtures by means of differential scanning calorimetry. The salting-out electrolyte NaCl expanded the Lα region toward higher temperatures and shrank the H1 region toward lower temperatures compared with the salt-free system. On the contrary, the salting-in electrolytes NaI and NaClO4 induced shrinkage of the Lα region and an expansion of the H1 phase. The influence of these salts on the mesophase regions was more pronounced for the Lα phase than for the H1 phase, and area of the Lα phase region decreased in the sequence of NaCl > none > NaI > NaClO4, consist with the Hofmeister series of the anions. This salt effect on the mesophase stability in aqueous nonionic surfactant mixture would be qualitatively interpreted in terms of the salt effect on the hydration of the polyoxyethylene chain in the surfactant molecules.

Because concern over endocrine disrupting reactions caused by chemicals to humans and animals is growing, a rapid and reliable screening assay for endocrine disrupting chemicals is required. We have developed an in vitro screening assay based on a hormone receptor mechanism using a surface plasmon resonance (SPR) sensor. The interaction between an estrogen receptor alpha (ER) and an estrogen response element (ERE) is monitored in real time, when ER is injected over the SPR sensor chip on which a DNA fragment containing ERE is immobilized. In the presence of a chemical with estrogenic activity, the ER-ERE interaction is enhanced and the kinetic parameters are altered. We have validated the assay in terms of its specificity, dose dependency, optimal reaction conditions and reproducibility. It has been shown that the assay is very reliable as a rapid and quantitative screening method to judge the estrogenic activities of chemicals.

Benzene is a potent human leukemogen but the mechanism underlying benzene-induced leukemia remains an enigma due to a number of questions regarding the requirement of extraordinarily long exposure, a relatively low incidence of leukemia for genotoxicity of metabolites and a narrow dose range for leukemogenicity over marrow aplasia (overdoses tend to result in marrow aplasia). Moreover, there were previous controversies as to whether the cell cycle is upregulated or suppressed by the benzene exposure. Subsequently, it was found that the cell cycle is suppressed, but how leukemia develops under such suppression of hemopoiesis remains to be clarified. These questions were fortunately resolved with much effort. Benzene exposure was found to induce the expression of p21, an interlocking counterdevice for cell cycle: due to p53 upregulation, thereby inducing the immediate suppression of the kinetics of hemopoietic progenitors followed by the prominent suppression of hemopoiesis. Intermittent benzene exposure (i.e., cessation of exposure during weekends, for example) allowed an immediate recovery from marrow suppression after terminating exposure, which induced continuous oscillatory changes in marrow hemopoiesis. Benzene-induced leukemia was chiefly due to such an oscillatory change in hemopoiesis, which epigenetically developed leukemia more than 1 year later. The mechanisms of benzene-induced leukemogenicity seem to differ between wild-type mice and mice lacking p53. For p53 knockout mice, DNA damage such as weak mutagenicity or chromosomal damage was retained, and such damage induced consequent activation of proto-oncogenes and related genes, which led cells to undergo further neoplastic changes. In contrast, for wild-type mice carrying the p53 gene, a marked oscillatory change in the cell cycle of the stem cell compartment seems to be important. Compatible and discriminative gene expression profiling between the p53 knockout mice and wild-type mice was observed after benzene exposure by microarray analyses.

The Alternatives to Carcinogenicity Testing Committee of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) conducted a large-scale, multinational collaborative research program to evaluate several genetically modified mouse assays for assessing the human carcinogenic potential of compounds. The data from this testing program have made an important contribution to the general understanding of how these models can be best applied in hazard identification; however, questions still exist regarding methodology and data interpretation. To address these issues, ILSI HESI hosted a February 2003 workshop on the Utility of Transgenic Assays for Risk Assessment. The purpose of this workshop was to reach an understanding of how data from genetically modified mouse models are viewed by different regulatory bodies in the pharmaceutical sector and, based on this understanding, to identify areas in which more experimental work may be needed to increase the utility of data derived from these assays. In the course of discussions, various data gaps related to model selection and protocol issues were identified. Based on the outcome of the workshop, various studies are proposed to provide data to improve the utility of currently available assays for cancer hazard identification and risk assessment purposes.

A 52-week study of oral-repeated-dose toxicity for the extraction powder of Gymnema sylvestre (GS), Indian-native genus, Metaplexis japonica, was conducted in both genders of Wistar rats. The rats were administered a graded dose of GS at 0.01, 0.10 and 1.00% of basal powder diet, along with a group fed solely with the basal powder diet without GS, for 52 weeks. General conditions were recorded daily. Body weights and food consumptions were recorded weekly up to 12 weeks, and thereafter at longer intervals. At 26 weeks, for an intermediate examination, and 52 weeks, for the final examination, animals were subjected to hematology, serum chemistry, and pathological examination. None of the animals died in the period up to 52 weeks. No exposure-related changes in body-weight, in the food consumption, in the hematological examinations, or in the serum biochemical examinations were recognized. No histopathological alterations were seen. Thus, it was concluded that there was no toxic effect in rats treated with GS at up to 1.00% in the diet for 52 weeks. The no-observable-effect level from this study is 1.00% GS, i.e., 504 mg/kg/day for male and 563 mg/kg/day for female as mean daily intake, for 52 weeks.

Regulatory and industrial scientists collaborated to publish a "points to consider" document regarding the safety assessment of biotechnology-derived pharmaceuticals in non-clinical studies in 2002 (Pharmaceutical Non-clinical Investigation Group, 2002). The collaboration team intended to clarify the interpretation of ICH-S6 guideline and furthermore share recent Japanese practices on this matter. However, the document was written in Japanese. Thus, we share here an English translation of the document so that non-native Japanese correctly understand the contents.

Oncostatin M (OSM) is a multifunctional cytokine that belongs to the interleukin 6 (IL-6) family. As OSM is expressed in adult as well as embryonic hematopoietic tissues, OSM has been considered to play a role in hematopoiesis. To uncover roles of OSM, we have generated mutant mice deficient in the OSM-specific receptor β subunit (OSMR). While OSMR–/– mice were healthy and fertile, hematologic analysis of OSMR–/– mice demonstrated that the numbers of peripheral erythrocytes and platelets were reduced compared with wild-type mice. Consistent with this, progenitors of erythroid and megakaryocyte lineages were reduced in OSMR–/– bone marrow (BM), suggesting that OSM is required for the maintenance of erythroid and megakaryocyte progenitor pools in BM. To investigate whether OSM acts on the hematopoietic progenitors directly or indirectly, we performed BM transplantation experiments. The OSMR–/– mice, engrafted with wild-type BM cells, failed to produce erythrocytic and megakaryocytic progenitors to the levels in wild-type mice, indicating that OSM affects hematopoietic microenvironments. On the other hand, erythrocytic and megakaryocytic progenitors were reduced in the wild-type mice reconstituted with OSMR–/– BM cells. Thus, OSM regulates hematopoiesis in vivo by stimulating stromal cells as well as hematopoietic progenitors, in particular megakaryocytic and erythrocytic progenitors.

The cloud point temperature, T(c), was investigated for aqueous solutions of poly(oxyethylene) alkyl ethers, C(n)E(m), and their mixtures. The experimental T(c)'s for single surfactant systems were analyzed according to the Flory-Huggins model for cloud point phenomenon, and the enthalpy and the entropy changes associated with the process of the separation of micellar solution into pure water and pure surfactant were estimated. It was found that the enthalpy-entropy compensation relationship holds for this process. The Flory-Huggins model was extended to the binary surfactant mixtures, and the expression of T(c) as a function of the composition was derived assuming the regular solution for mixed micelles. The experimental results of T(c) obtained for mixtures of C(n)E(m) were well reproduced by the model calculation. Discussion is given concerning the interaction parameters of different surfactant species in mixed micelles determined by this model calculation.

The pseudo-binary T-X phase diagrams for aqueous mixtures of polyethylene glycol alkyl ethers with chain length distribution were constructed by means of differential scanning calorimetry and were compared with the binary phase diagrams for the aqueous mixtures of surfactants with a homogeneous chain length distribution. The surfactants with chain length distribution were prepared by mixing CnEm (n = 10, 14, and m = 5 - 8) in appropriate molar ratio to provide average chain lengths of n = 12 and m = 6 or 7. The regions of mesophases appearing in the aqueous mixtures of mixed surfactants were essentially the same as those in aqueous mixtures of pure surfactants with chain lengths corresponding to the average chain lengths of the mixed surfactants. This suggests that the aqueous phase behavior of the POE-type surfactants with chain length distribution is determined by the average chain length in both the POE chain and the hydrocarbon chain so long as the chain length difference among the components is not so large.

A hormonally active compound was first identified in the book Silent Spring by Rachel Carson in 1962, implicating the effect of pesticides such as DDT and the derivatives. Nearly four decades later, the book Our Stolen Future by Theo Colborn et al., and other pertinent publications have revisited and broadened the issue regarding a variety of possible chemicals and the area exposed. Translation and publication became available in Japan within the last four years. Since then, Japan joined the member countries involved in the global issue of endocrine disruptors, the "environmental hormone". Although a significant number of chemicals possessing a hormone-like action have been recognized for many years, and the action of their biological plausibility related to the receptor-mediated effects strongly suggests possible human effects comparable to hormonal changes in wildlife, little is known about evidences or adversities in experimental animals and humans. The most essential key to resolving these dilemmas may be to understand the mechanism of actions (i.e., a possible low-dose issue). In other words, the mechanism at the low-dose effect may be resolved simultaneously by the mechanism of three major questions linked to the low-dose issue; namely, threshold, possible oscillation, and additive and/or synergistic action.

This workshop was convened to address common issues and concerns associated with risk management of endocrine-disrupting chemicals (EDCs). The talks described the tools and policies for key Japanese, Australian, German, and U.S. regulatory agencies. The agencies participating in the workshop were responsible for the regulation of various substances including: chemicals, pesticides, environmental contamination, pharmaceuticals, and food additives. The panel also described the role of the Organization for Economic Cooperation and Development (OECD) in standardizing the tools and validation of testing and screening methods. The panel also included nongovernmental organizations presenting the views of the World Wildlife Fund, and the chemical industry from industrialized nations; each organization described its concerns and proposed approaches to risk management of EDCs. This summary highlights the most important areas of common points of view of government, industry, and environmentalists. We also try to identify issues upon which viewpoints diverge.

Surface tension measurements were carried out for aqueous solutions of polyoxyethylene oleyl and stearyl ethers with average number of oxyethylene units of 20 (abbreviated as POEOl and POESt, respectively) in order to examine the effect of introducing a double bond into hydrocarbon chain on the surface activity of the surfactants. The overall features of surface chemical properties of these two surfactant solutions are similar to each other, although the surface tension and the critical micelle concentration (CMC) are slightly higher while the amount of surface adsorption is slightly smaller for POEOl compared with those for POESt. Thermodynamic parameters of micelle formation derived from the temperature dependence of the CMC are also close to each other for the two surfactant species. These results demonstrate that the effect of double bond in the surfactant hydrocarbon chain on the surface activity is rather small at least for nonionic surfactant species with relatively long polyoxyethylene chain.

近年ヒト, 野生動物の内分泌系を撹乱する化学物質の存在が懸念されており, これらの化学物質の作用機構として, 内分泌系を制御する種々のホルモンレセプターと化学物質との相互作用が指摘されている. 著者らは内分泌撹乱化学物質の迅速なin vitro スクリーニング法としてホルモンレセプターの作用機構に焦点をあてた表面プラズモン共鳴高速分析法を構築した. 表面プラズモン共鳴センサーのセンサーチップ上にエストロゲンレセプターのホルモン応答配列DNA (ERE) を固定化し, エストロゲンレセプターα (ER) を添加するとERとEREの相互作用を非標識でリアルタイムに測定することができる. この系でERを様々な化学物質と共存させると, ERとEREの結合・解離の反応速度が変化する. このアッセイ法について特異性, 用量依存性, 至適条件及び系の再現性を検討した. その結果, この方法はある化学物質がエストロゲン様作用を有するかどうかを迅速かつ定量的に判定するスクリーニング法として有効であることが示された.

Cytokeratin (CK) 20 expression is an independent prognostic factor of poorly differentiated adenocarcinoma (PDA) of the colon and rectum. We aimed to investigate the mechanism of its involvement through a clinicopathological study.We analyzed 156 surgically resected PDAs, which were sub-classified as solid type (Por1) showing expansive growth, or non-solid type (Por2) showing infiltrative growth. Associations of CK20 expression with morphological features and molecular markers were analyzed.CK20(+) PDA (n=91) was associated with more advanced disease stage and unfavorable prognosis compared with CK20(-) PDA (n=65). Pathologically, CK20(+) PDA was significantly associated with p53 overexpression, Por2, abundant fibrous stroma, and stepwise de-differentiation, while CK20(-) PDA was significantly associated with mismatch repair deficiency, Por1, sparse fibrous stroma, and de novo histogenesis.CK20 expression in PDA is closely associated with invasive histological features, providing prognostic significance, and may also point to a specific histogenetic pathway.

Mesp1 and Mesp2 encode bHLH-type transcription factors, Mesp1 and Mesp2, respectively. The expression of both genes is observed in the nascent mesoderm, and subsequently in the rostral presomitic mesoderm. To determine the regulatory mechanism for gene expression, we attempted to identify enhancer elements by transient transgenic analysis. At least two enhancers, which are responsible for the expression of the two genes in the early mesoderm (early mesodermal enhancer, EME) and the presomitic mesoderm (PSM enhancer, PSME), and one suppressor, which is responsible for the rostrally restricted expression in the presomitic mesoderm, were identified. Deletion studies of these enhancer elements indicate that either gene may use the same enhancer for early mesoderm development, whereas both genes may utilize separate enhancers to regulate their expression in the presomitic mesoderm.