Presence of mast cell precursors in the yolk sac of mice

Concentration of mast-cell precursors in hematopoietic tissues of mouse embryos was evaluated by a limiting dilution method. Cells from yolk sacs, livers, and bodies of (WB x C57BL/6)F1 (hereafter called WBB6F1)- +/+ embryos were injected directly into the skin of adult WBB6F1-W/Wv mice which were genetically depleted of tissue mast cells. Concentration of mast-cell precursors was calculated from the proportion of injection sites at which mast cells did not appear. Since the concentration of mast-cell precursors in the yolk sac was about 30 times as great as that of embryonic body at Day 9.5 of the pregnancy, the mast-cell precursors seemed to be generated within the yolk sac. The concentration in the yolk sac reached the maximum level at Day 11, and then dropped markedly at Day 13. In contrast, mast-cell precursors increased from Day 11 to Day 15 in the fetal liver. As a result, the concentration of 11-day yolk sacs was comparable to that of 15-day fetal liver. Although intravenous injection of 15-day fetal liver cells (2 x 10(6)) rescued the general mast-cell depletion of WBB6F1-W/Wv mice, the intravenous injection of the same number of 11-day yolk sac cells did not rescue it. In contrast with fetal livers, yolk sacs scarcely contained hematopoietic stem cells which were measured by spleen colony formation. Therefore, the mast-cell precursors of the yolk sac may not originate from such stem cells.     

An improved method based on the Porter-Silber reaction for determining 17-hydroxycorticosteroids in urine

This paper describes a highly specific method for determining urinary 17-hydroxycorticosteroids, which has been developed by (i) changing the composition of the Porter-Silber reagent and (ii) removing contaminants interfering with the color reaction by addition of sodium bisulfite to β-glucuronidase-hydrolyzed urine before extraction with solvent. For a reference method the Norymberski-Riondel (J. K. Norymberski and A. Riondel 1970, Biochem. J. 120, 493–498) gas chromatography (glc) was used: Correlation coefficient between the present method and GLC = 0.988, deviation from the theoretical regression LINE = 6.8%, and coefficient of SIMILARITY = 0.56. These results are much better than those obtained by I. Ernest, B. Håkansson, J. Lehmann, and B. Sjögren (1964, Acta Endocrinol. 46, 552–562) for the original Porter-Silber method in comparison with the chromatographic measurement of grouped and individual steroids.     

Adenovirus infection induces HuR relocalization to facilitate virus replication

HuR is an RNA-binding protein of the embryonic lethal abnormal vision (ELAV) family, which binds to the AU-rich element (ARE) in the 3′-untranslated region (UTR) of certain mRNAs and is involved in the nucleo-cytoplasmic export and stabilization of ARE-mRNAs. The cytoplasmic relocalization of ARE-mRNAs with several proteins such as HuR and pp32 increases in cells transformed by the adenovirus oncogene product E4orf6. Additionally, these ARE-mRNAs were stabilized and acquired the potential to transform cells. Although, the relocalization of HuR and the stabilization of ARE-mRNAs are crucial for cell transformation, evidence regarding the relationship of HuR and ARE-mRNAs with adenovirus replication is lacking. In this report, we demonstrate that adenovirus infection induces the relocation of HuR to the cytoplasm of host cells. Analysis using the luciferase-ARE fusion gene and the tetracycline (tet)-off system revealed that the process of stabilizing ARE-mRNAs is activated in adenovirus-infected cells. Heat shock treatment or knockdown-mediated depletion of HuR reduced adenovirus production. Furthermore, expression of ARE-including viral IVa2 mRNA, decreased in HuR-depleted infected cells. These results indicate that HuR plays an important role in adenovirus replication, at least in part, by up-regulating IVa2 mRNA expression and that ARE-mRNA stabilization is required for both transformation and virus replication.     

MK-8776, a novel Chk1 inhibitor,exhibits an improved radiosensitizing effect compared to UCN-01 by exacerbating radiation-induced aberrant mitosis

Checkpoint kinase 1 (Chk1) is an evolutionarily conserved serine/threonine kinase that plays an important role in G₂/M checkpoint signaling. Here, we evaluate the radiosensitizing effects of a novel selective Chk1 inhibitor MK-8776, comparing its efficacy with a first-generation Chk1 inhibitor UCN-01, and attempt to elucidate the mechanism of radiosensitization. In a clonogenic survival assay, MK-8776 demonstrated a more pronounced radiosensitizing effect than UCN-01, with lower cytotoxicity. Importantly, radiosensitization by MK-8776 can be achieved at doses as low as 2.5 Gy, which is a clinically applicable irradiation dose. MK-8776, but not UCN-01, exacerbated mitotic catastrophe (MC) and centrosome abnormalities, without affecting repair kinetics of DNA double strand breaks. Furthermore, live-cell imaging revealed that MK-8776 significantly abrogated the radiation-induced G₂/M checkpoint, prolonged the mitotic phase, and enhanced aberrant mitosis. This suggests that Chk1 inhibition by MK-8776 activates a spindle assembly checkpoint and increases mitotic defects in irradiated EMT6 cells. In conclusion, we have shown that, at minimally toxic concentrations, MK-8776 enhances radiation-induced cell death through the enhancement of aberrant mitosis and MC, without affecting DNA damage repair.     

Hyperinnervation improves Xenopus laevis limb regeneration

Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors.     

A keratin of fetal skin is reexpressed in human keratinocytes transformed by SV40 virus or treated with the tumor promoter TPA

SV40-transformation as well as treatment with tumor promoters produce alterations in morphology, differentiation and keratinization of human keratinocytes. Two cell lines of SV40-transformed keratinocytes and primary cultures of keratinocytes treated with the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) were found to contain an additional protein of 52.5 kD molecular weight (MW). This protein was identified by its reactivity with the monoclonal antibody TROMA-I as being keratin no. 8, a keratin normally present only in simple epithelia. Since this keratin is present in fetal epidermis but disappears gradually when fetal skin becomes multilayered after week 13 of development (Moll et al., Differentiation 23 (1982) 170. [23]), it suggests that SV40 virus and TPA are able to induce in human keratinocytes the reexpression of fetal characters.