Hes1 suppresses acute myeloid leukemia development through FLT3 repression.

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix-loop-helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL-AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL-AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL-AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL-AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL-AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

Integrin α3 is overexpressed in glioma stem-like cells and promotes invasion.

BACKGROUND: Glioma stem-like cell (GSC) properties are responsible for gliomagenesis and recurrence. GSCs are invasive but its mechanism remains to be elucidated. Here, we attempted to identify the molecules that promote invasion in GSCs. METHODS: Neurospheres and CD133⁺ cells were collected from glioblastoma (GBM) specimens and glioma cell lines by sphere-formation method and magnetic affinity cell sorting, respectively. Differential expression of gene candidates, its role in invasion and its signaling pathway were evaluated in glioma cell lines. RESULTS: Neurospheres from surgical specimens attached to fibronectin and laminin, the receptors of which belong to the integrin family. Integrin α3 was overexpressed in CD133⁺ cells compared with CD133⁻ cells in all the glioma cell lines (4 out of 4). Immunohistochemistry demonstrated the localisation of integrin α3 in GBM cells, including invading cells, and in the tumour cells around the vessels, which is believed to be a stem cell niche. The expression of integrin α3 was correlated with migration and invasion. The invasion activity of glioma cells was linked to the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. CONCLUSION: Our results suggest that integrin α3 contributes to the invasive nature of GSCs via ERK1/2, which renders integrin α3 a prime candidate for anti-invasion therapy for GBM.

Biological clock dysfunction exacerbates contact hypersensitivity in mice.

BACKGROUND: Immediate-type skin allergic reactions, such as passive cutaneous anaphylactic reaction, are associated with circadian rhythm, but the role of circadian mechanisms on delayed-type skin allergic reactions, such as contact hypersensitivity (CHS), remains uncertain. In mice, CHS, a T-cell-mediated immune response, is a classic model of human allergic contact dermatitis. OBJECTIVES: We investigated whether biological clock dysfunction affects CHS pathogenesis in CLOCK mutant mice compared with wild-type (WT) mice. METHODS: Mice were treated with 2,4,6-trinitro-1-chlorobenzene (TNCB) on the abdominal skin on day 0 (sensitization) and then treated with TNCB on the ears on day 5 (challenge). RESULTS: We found that biological clock dysfunction resulted in severe inflammation. Ear swelling, serum immunoglobulin E level and mast cell number were significantly increased in CLOCK mutant mice compared with WT mice. These results provide evidence that CLOCK mutation promotes the T-helper type 2 immune response and exacerbates CHS. Corticosterone has a protective effect on CHS. The serum corticosterone level lost rhythmicity and showed a decreased daily level in CLOCK mutant mice compared with WT mice, supporting the exacerbating effect of CLOCK mutation on CHS. Adrenalectomy markedly worsened TNCB-induced CHS in WT mice but not in CLOCK mutant mice. In addition, dramatic dexamethasone-induced protection of CHS was observed in CLOCK mutant mice compared with WT mice. CONCLUSIONS: The present results suggest that circadian rhythm might be an important factor in the regulation of CHS via corticosterone rhythmicity and/or level.

Effect of temperature on the development of Steinernema carpocapsae and Steinernema feltiae (Nematoda: Rhabditida) in liquid culture.

For commercial use of the entomopathogenic nematodes Steinernema carpocapsae and Steinernema feltiae in biological control of insect pests, they are produced in liquid culture on artificial media pre-incubated with their symbiotic bacteria Xenorhabdus nematophila and Xenorhabdus bovienii, respectively. After 1 day of the bacterial culture, nematode dauer juveniles (DJs) are inoculated, which recover development. The adult nematodes produce DJ offspring, which are harvested and can be sprayed. This study determined optimal temperatures to obtain high DJ progeny within a short process time. Temperatures assessed were 23 degrees C, 25 degrees C, 27 degrees C, and 29 degrees C for S. carpocapsae and 20 degrees C, 23 degrees C, 25 degrees C, and 27 degrees C for S. feltiae. The recovery of inoculated DJs was hardly affected and was reduced only in S. carpocapsae at 29 degrees C. The fecundity (eggs in uterus) in S. carpocapsae reached a maximum at 27 degrees C; whereas, maximum yields were recorded at 25 degrees C. For both Steinernema spp., highest DJ densities were obtained after 15 days incubation at 25 degrees C. Optimal culture temperature for both nematode species is 25 degrees C. S. carpocapsae was more sensible to suboptimal temperature than S. feltiae. Results on total DJ density and DJ proportion of the total nematode population were more variable at non-optimal temperature condition for S. carpocapsae than for S. feltiae. Suboptimal culture temperature also reduced DJ infectivity.

Influence of cell density and phase variants of bacterial symbionts (Xenorhabdus spp.) on dauer juvenile recovery and development of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida).

The rhabditid nematodes Steinernema carpocapsae and Steinernema feltiae are used in biological control of insect pests. Mass production is done in liquid culture media pre-incubated with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, before nematode dauer juveniles (DJs) are inoculated. As a response to food signals produced by the bacterial symbionts, the DJs exit from the developmentally arrested dauer stage (they recover development) and grow to adults, which produce DJ offspring. Variable DJ recovery after inoculation often causes process failure due to non-synchronous population development and low numbers of adult nematodes. This contribution investigated the influence of the bacterial cell density on DJ recovery and development to adults. At higher density of 10(10) bacterial cells ml(-1), a higher percentage of DJ recovery was induced, and adults occurred earlier in both Steinernema spp. than at lower density of 10(9) and 10(8) cells ml(-1). Xenorhabdus symbionts produce phase variants. Recovery in bacteria-free supernatants was lower than in supernatants containing bacterial cells for both primary and secondary phase Xenorhabdus spp. and lower in secondary than in primary phase supernatants or cell suspensions. In general, recovery was lower for Steinernema feltiae and the time at which 50% of the population had recovered after exposure to the food signal was longer (RT(50) = 17.1 h) than for Steinernema carpocapsae (RT(50) = 6.6 h). Whereas >90% S. carpocapsae DJs recovered in hemolymph serum of the lepidopteran insect Galleria mellonella, recovery of S. feltiae only reached 31%. Penetration into a host insect prior to exposure to the insect's food signal did not enhance DJ recovery. Consequences for liquid culture mass production of the nematodes and differences between species of the genera Steinernema and Heterorhabditis are discussed.

The effect of segregation of flowering time on fine-scale spatial genetic structure in an alpine-snowbed herb Primula cuneifolia.

The flowering phenology of alpine-snowbed plants varies widely depending on the time of snowmelt. This variation may cause spatial and temporal heterogeneity in pollen dispersal, which in turn may influence genetic structure. We used spatial autocorrelation analyses to evaluate relative effect of segregation in flowering time and physical distance on fine-scale spatial genetic structure (SGS) of a snowbed herb Primula cuneifolia sampled in 10-m grids within a continuous snow patch (110 x 250 m) using nine allozyme loci. Although the individual flower lasts for

Seasonal changes in pollinator activity influence pollen dispersal and seed production of the alpine shrub Rhododendron aureum (Ericaceae).

In alpine ecosystems, microscale variation in snowmelt timing often causes different flowering phenology of the same plant species and seasonal changes in pollinator activity. We compared the variations in insect visitation, pollen dispersal, mating patterns, and sexual reproduction of Rhododendron aureum early and late in the flowering season using five microsatellites. Insects visiting the flowers were rare early in the flowering season (mid-June), when major pollinators were bumblebee queens and flies. In contrast, frequent visitations by bumblebee workers were observed late in the season (late July). Two-generation analysis of pollen pool structure demonstrated that quality of pollen-mediated gene flow was more diverse late in the season in parallel with the high pollinator activity. The effective number of pollen donors per fruit (N(ep)) increased late in the season (N(ep) = 2.2-2.7 early, 3.4-4.4 late). However, both the outcrossing rate (t(m)) and seed-set ratio per fruit were smaller late in the season (t(m) = 0.89 and 0.71, seed-set ratio = 0.52 and 0.18, early and late in the season, respectively). In addition, biparental inbreeding occurred only late in the season. We conclude that R. aureum shows contrasting patterns of pollen movement and seed production between early and late season: in early season, seed production can be high but genetically less diverse and, during late season, be reduced, possibly due to higher inbreeding and inbreeding depression, but have greater genetic diversity. Thus, more pollinator activity does not always mean more pollen movement.

Landscape genetics of alpine-snowbed plants: comparisons along geographic and snowmelt gradients.

The genetic structure of three snowbed-herb species (Peucedanum multivittatum, Veronica stelleri, and Gentiana nipponica) was analyzed using allozymes across nine populations arranged as a matrix of three snowmelt gradients x three geographic locations within 3 km in the Taisetsu Mountains, northern Japan. Phenologically asynchronous populations are packed within a local area in alpine snowbeds, because flowering season of alpine plants depends strongly on the timing of snowmelt. Moderate genetic differentiation was detected among local populations in every species (FST=0.03-0.07). There was a significant correlation between the geographic distance and genetic distance in the P. multivittatum populations, but not in the V. stelleri and G. nipponica populations. On the other hand, a significant correlation between the phenological distance caused by snowmelt timing and genetic distance was detected in the V. stelleri and G. nipponica populations, but not in the P. multivittatum populations. The snowmelt gradient or geographic separation influenced hierarchical genetic structure of these species moderately (FRT <0.04). Restriction of gene flow due to phenological separation and possible differential selection along the snowmelt gradient may produce genetic clines at microgeographic scale in these species.

Effect of dietary conjugated linoleic acid on the in vivo growth of rat hepatoma dRLh-84.

We examined the effect of dietary conjugated linoleic acid (CLA) on the growth of injected hepatoma dRLh-84 in Donryu rats. After experimental diets containing 0% or 2% CLA were given to male Donryu rats for 3 wk, dRLh-84 cells were injected into the left lobe of the hepatic capsule, and the experimental diet was continued. The cells formed a solid tumor > or = 1 wk after the injection, and thereafter the tumor grew with feeding duration. In a morphological study, this tumor appeared to be a low-differentiated hepatoma, and there was no remarkable difference in the morphology of the tumor between 0% and 2% CLA groups. Tumor weight was significantly higher in the 2% CLA group than in the 0% CLA group throughout the feeding period after the injection. Serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activities were significantly higher in 2% CLA-injected rats than in 0% CLA-injected rats at 3 wk after the injection. CLA upregulated acyl-CoA oxidase activity, especially 1 wk after the injection. However, dietary CLA did not activate carnitine palmitoyl transferase II, which is a rate-limiting enzyme in the mitochondrial beta-oxidation pathway. Natural killer cell activity in the spleen tended to be higher in injected rats, but a significant effect of dietary CLA was not recognized. Serum interferon-gamma and tumor necrosis factor-alpha levels were higher in injected than in sham rats. Moreover, these levels were higher in 2% CLA groups than in the respective 0% CLA groups.

DNA damage-induced activation of p53 by the checkpoint kinase Chk2.

Chk2 is a protein kinase that is activated in response to DNA damage and may regulate cell cycle arrest. We generated Chk2-deficient mouse cells by gene targeting. Chk2-/- embryonic stem cells failed to maintain gamma-irradiation-induced arrest in the G2 phase of the cell cycle. Chk2-/- thymocytes were resistant to DNA damage-induced apoptosis. Chk2-/- cells were defective for p53 stabilization and for induction of p53-dependent transcripts such as p21 in response to gamma irradiation. Reintroduction of the Chk2 gene restored p53-dependent transcription in response to gamma irradiation. Chk2 directly phosphorylated p53 on serine 20, which is known to interfere with Mdm2 binding. This provides a mechanism for increased stability of p53 by prevention of ubiquitination in response to DNA damage.

Determination of left/right asymmetric expression of nodal by a left side-specific enhancer with sequence similarity to a lefty-2 enhancer.

The nodal gene is expressed on the left side of developing mouse embryos and is implicated in left/right (L-R) axis formation. The transcriptional regulatory regions of nodal have now been investigated by transgenic analysis. A node-specific enhancer was detected in the upstream region (-9.5 to -8.7 kb) of the gene. Intron 1 was also shown to contain a left side-specific enhancer (ASE) that was able to direct transgene expression in the lateral plate mesoderm and prospective floor plate on the left side. A 3. 5-kb region of nodal that contained ASE responded to mutations in iv, inv, and lefty-1, all genes that act upstream of nodal. The same 3. 5- kb region also directed expression in the epiblast and visceral endoderm at earlier stages of development. Characterization of deletion constructs delineated ASE to a 340-bp region that was both essential and sufficient for asymmetric expression of nodal. Several sequence motifs were found to be conserved between the nodal ASE and the lefty-2 ASE, some of which appeared to be essential for nodal ASE activity. These results suggest that similar transcriptional mechanisms underlie the asymmetric expression of nodal and of lefty-2 as well as the earlier expression of nodal in the epiblast and endoderm.

Distinct transcriptional regulatory mechanisms underlie left-right asymmetric expression of lefty-1 and lefty-2.

Both lefty-1 and lefty-2 genes are expressed on the left side of developing mouse embryos and are implicated in left-right (L-R) axis formation. With the use of transgenic analysis, the transcriptional regulatory regions of these genes responsible for their L-R asymmetric expression have now been investigated. The 9.5-kb upstream region of lefty-1 and the 5.5-kb upstream region of lefty-2 reproduced the expression pattern of the corresponding gene. Examination of deletion constructs revealed the presence of a left side-specific enhancer (ASE) that is essential and sufficient for lefty-2 asymmetric expression. In contrast, the asymmetric expression of lefty-1 was shown to be determined by a combination of bilateral enhancers and a right side-specific silencer (RSS). The 9. 5-kb region of lefty-1 and the 5.5-kb region of lefty-2 responded to iv and inv, upstream genes of lefty-1 and lefty-2. The regulation of lefty-2 by iv and inv was mediated by ASE. These results suggest that, in spite of the similarities between lefty-1 and lefty-2, different regulatory mechanisms underlie their asymmetric expression.

Quantitative detection of hepatitis B virus by transcription-mediated amplification and hybridization protection assay.

We have developed a sensitive and quantitative assay using transcription-mediated amplification and hybridization protection assay for the detection of hepatitis B virus (HBV) DNA in serum. The transcription-mediated amplification was carried out in a single tube. The hybridization protection assay was carried out in a microtiter plate with two probes with different specific activities to obtain a broad detection range. As a result, the assay had a detection range of 5 x 10(3) to 5 x 10(8) genome equivalents (GE)/ml and good quantitative accuracy on a logarithmic scale. A moderately sized manual assay run can be completed within 5 h. Measurements of the amounts of HBV DNA in clinical samples by the assay showed the amounts under various disease conditions to be widely distributed (more than 5 logs, from approximately 5 x 10(3) to 5 x 10(8) GE/ml). It was also shown that the amount of HBV DNA in one chronic hepatitis patient varied widely, with a range of more than 5 logs during long-term monitoring. Our assay has the potential to be used to monitor and determine the prognosis of HBV patients and carriers, especially during interferon treatment.

Clinically applicable bulk isolation of blood CD34+ cells for autografting in children.

CD34+ cells were purified in bulk from apheresis-collected cells of children with cancer using monoclonal antibody (MoAb) and magnetic beads (Baxter ISOLEX system). To improve the purity of the final product for possibly better tumor cell purging and to make the manufacturer's original procedure more cost-effective, we incubated the cells for 30 min with l-phenylalanine methylester hydrochloride (PME) to reduce the cell number by removing contaminating granulocytes and monocytes in the initial step before incubation with MoAb. Our modification prevented nonspecific interactions between MoAb and magnetic beads, and thereby saved expensive materials for purification. A total of 40 purifications were performed with samples containing a mean of 3.1 x 10(9) blood cells mobilized from 15 children by chemotherapy plus granulocyte colony-stimulating factor (G-CSF). The entire purification procedure, from the end of apheresis to storage, was completed within 5h. After incubation with PME and double-layered (40/60%) Percoll separation, the number of CD34+ cells was reduced to 48+/-29%, which suggests the possibility that half of the CD34+ cells in the inoculum were nonclonogenic in the hematopoietic progenitor assay. PME/Percoll-treated cells were then subjected to a final isolation procedure with MoAb according to the manufacturer's suggestions, and 52+/-42% and 32+/-22%, respectively, of the CFU-GM and CD34+ cells present in the initial bag inoculums were recovered. The recovery rates were, respectively, 54% and 67%, when the calculation was limited to the isolation procedure with MNoAb. The purity of isolated CD34+ cells and the plating efficiency in methylcellulose culture were, respectively, 77+/-24% and 33+/-13%. Fourteen children were subsequently autografted with purified CD34+ cells after marrow ablative chemotherapy. The median number of days to achieve an ANC of 0.5 x 10(9)/l was 12 and that to achieve a platelet count of 50 x 10(9)/l was 22.5, which were comparable to those in our historical group of 55 patients who underwent transplant with unmanipulated blood cells (13 and 16 days). These results suggest that our modified purification procedure with PME is useful for the initial reduction of cell numbers to save costly materials, and that cells isolated by this procedure can be directly used in clinical transplantation procedures.

Overexpression of C-terminal Src kinase homologous kinase suppresses activation of Lyn tyrosine kinase required for VLA5-mediated Dami cell spreading.

The Csk homologous kinase (Chk), which is co-expressed with C-terminal Src kinase (Csk) in hematopoietic cells, negatively regulates Src family kinases in vitro with selectivity toward Lyn but not c-Src in platelets. To explore the role of Src family kinases in hematopoietic cell adhesion, we overexpressed Chk in the megakaryocytic cell line Dami and established clones exhibiting a 10-fold increase in the amount of Chk. Overexpression of Chk was found to suppress VLA5 integrin-mediated cell spreading, but not cell attachment, throughout fibronectin (FN) stimulation. Deletion and point mutagenesis analyses of Chk showed that this suppression was dependent upon both the SH3 domain, which is responsible for membrane anchoring, and kinase activity. FN-induced cell spreading accompanied a sustained increase in Lyn activity with coincidental kinetics and the activation of Lyn was also suppressed by overexpression of Chk but not a Chk mutant lacking the SH3 domain. Expression of a truncated Lyn mutant lacking the kinase domain inhibited both cell spreading and Lyn activation upon stimulation with FN. These results suggest that sustained activation of Lyn, which is regulated by membrane-anchored Chk, plays a crucial role in VLA5-mediated cell spreading but not cell attachment to a FN substrate.

Translocation of the Csk homologous kinase (Chk/Hyl) controls activity of CD36-anchored Lyn tyrosine kinase in thrombin-stimulated platelets.

Chk/Hyl is a recently isolated non-receptor tyrosine kinase with greatest homology to a ubiquitous negative regulator of Src family kinases, Csk. To understand the significance of co-expression of Chk and Csk in platelets, we examined the subcellular localization of each protein. Chk, but not Csk, was completely translocated from the Triton X-100-soluble to the Triton X-100-insoluble cytoskeletal fraction within 10 s of thrombin stimulation. Chk and Lyn, but not Csk and c-Src, co-fractionated in the higher density lysate fractions of resting platelets, with Chk being found to localize close to CD36 (membrane glycoprotein IV)-anchored Lyn. The kinase activity of co-fractionated Lyn was suppressed 3-fold. In vitro phosphorylation assays showed that Chk suppressed Lyn activity by phosphorylating its C-terminal negative regulatory tyrosine. Upon stimulation of platelets with thrombin, the rapid and complete translocation of Chk away from Lyn caused concomitant activation of Lyn. This activation was accompanied by dephosphorylation of Lyn at its C-terminal negative regulatory tyrosine in cooperation with a protein tyrosine phosphatase. These results suggest that Chk, but not Csk, may function as a translocation-controlled negative regulator of CD36-anchored Lyn in thrombin-induced platelet activation.

Analysis of CSK homologous kinase (CHK/HYL) in hematopoiesis by utilizing gene knockout mice.

CHK/HYL is a non-receptor tyrosine kinase that belongs to CSK (C-terminal Src kinase) family. Northern blotting and RT-PCR analyses showed that CHK/HYL was expressed in large spectrum of hematopoietic cells except for erythroid cells and brain. To explore the function of CHK/HYL in hematopoietic cells, we generated CHK/HYL deficient mice. The mutant mice were apparently normal and fertile, while CSK knockout mice died until E11.5 from a defect in the neural tube formation. Hematological observations including blood counts and FACS analysis showed no significant abnormalities in CHK/HYL mutant mice. CHK/HYL did not affect the activity of Src, Hck, and Fgr in cultured bone marrow cells, although CSK negatively regulates Src family kinases. These results suggest that CHK/HYL might not have the same function as CSK.