Kazuko Tsuneoka

Induction of the Expression of the Interleukin-1β Gene in Mouse Spleen by Ionizing Radiation

Spleen cells freshly isolated from normal mice were irradiated with 20 Gy X rays in culture. Northern blot hybridizations revealed that expression of the interleukin-1 beta (IL-1 beta) gene was induced immediately after irradiation and was increased for 2 h thereafter. Dibutyryl cyclic AMP also caused a persistent expression of the IL-1 beta gene, although it differed from X rays in that it coincidentally induced expression of the c-fos gene, which was not induced by X rays. Activation of either protein kinase C or calmodulin also induced early expression of both IL-1 beta and c-fos. Myeloid cells collected from the spleen of mice with granulocytic leukemia were X-irradiated in culture as above. The leukemia cells responded to X rays as well as to other stimuli in the same manner as the spleen cells, except that IL-1 beta mRNA was no longer detected 30 min after irradiation while c-fos was detectable for 2 h. When the leukemia cells were irradiated twice with a 3-h interval between irradiations, the second irradiation led to prolonged expression of IL-1 beta without inducing c-fos expression. These results suggest that ionizing radiation elicits early expression of the IL-1 beta gene through a mechanism that does not involve protein kinase C or A, or the transcription factor, c-fos. Whole-body irradiation of mice with 50 Gy 137Cs gamma rays also induced IL-1 beta expression in spleen but not in bone marrow or liver, although there was a delay of several hours before it was amply expressed. Furthermore, a delay as long as 24 or 72 h was observed when the radiation dose was reduced to 8.5 or 4 Gy. The results of this in vivo study suggest that the rapidity of expression of the IL-1 beta gene is dependent on the dose of radiation, and that the cells in the body cannot respond to radiation as rapidly as cells in culture.

Radioprotection of mice by a single subcutaneous injection of heat-killed Lactobacillus casei after irradiation.

Treatment of whole-body gamma-irradiated mice with a preparation of Lactobacillus casei (LC 9018) immediately after irradiation caused a sustained increase in serum colony-stimulating activity which was followed by an enhanced repopulation of granulocyte-macrophage colony-forming cells in the femoral marrow and spleen. Numbers of blood leukocytes, erythrocytes, and platelets were increased earlier in the treated mice than in the controls, and the survival rate was elevated significantly. The radioprotective effect was dependent on the dose of LC 9018 as well as on the dose of radiation. These results demonstrate the value of LC 9018 for the treatment of myelosuppression after radiotherapy or radiation accidents.

The roles of hetero atoms in solvolytic reactions—II: Transannular participation by neighbouring sulphur atom

Abstract Esters of sulphur-containing 5- and 6-membered heterocycles, tetrahydro-3- and 4-thiopyranol and their 3- and 4-methyl, phenyl derivatives, and tetrahydro-2-thiophenemethanol, were synthesized. The solvolysis of β-thioesters in 80% aqueous acetone resulted in strong transannular S-participation and the products of solvolysis substantiated formation of bicyclic episulphonium ion intermediates. Such participation is considered to be present even in a tertiary system. On the other hand, γ-thioesters were solvolysed without any transannular S-participation,

2-Aminoindoles : Preparation from 2-indolinethiones, tautomerism and autoxidation

Abstract 2-Aminoindoles (2 and 4) have been prepared by the reaction of 2-indolinethiones (1) with primary and secondary aliphatic amines. The reaction of 1b with ethyleneimine produced the S-alkylated product 5, and with phenylhydrazine compound 9. The reaction 1b with aniline and 10 with piperidine failed to give an 2-aminoindole derivative or 12. The UV and NMR spectra of the free base 2 shows the presence of a tautomeric mixture of 13 and 14 in bases 2c, 2g, 2h and 2i. The free base 2b in CDCl3 exists substantially in the indolenine form 15. The free bases of 2b, 2g, 2h and 2i are susceptible to autoxidation to 3-oxo derivatives (19, 21, 22 and 23).

A sialoglycoprotein-stimulating proliferation of granulocyte-macrophage progenitors in mouse-bone marrow cell cultures

Since the method was established for culturing macrophage-granulocyte progenitor cells in vitro [ 1,2] , a considerable amount of work has been done to elucidate the molecular properties of the hormonelike substance (CS-factor) which is required for proliferation of progenitor cells in culture. A highly purified preparation was obtained by Guez and Sachs [3] from culture medium of a line of mouse cells (EI-cells). According to these workers, the substance is a simple protein which requires a low molecular weight cofactor for its action. In contrast, another CS-factor which was more recently purified by Stanley et al. [4] from human urine, is a sialoglycoprotein and requires no cofactors in an assay system similar to that of Guez and Sachs. The present paper reports evidence that a CSfactor which we have partially purified from the culture medium of a subline of mouse Lcells is a sialoglycoprotein. It seems, however, that the sialic acid residue on the protein is not essential for its action, because neuraminidase treatment increased the protein’s isoelectric point from 3.5-5.4 but did not significantly change its activity. Furthermore, both sialoand asialo-CS-factor required no cofactors in our assay system.

Increased expression of intracisternal A-particle RNA in regenerated myeloid cells after X irradiation in C3H/He inbred mice.

Abstract Ishihara, H., Tanaka, I., Furuse, M. and Tsuneoka, K. Increased Expression of Intracisternal A-Particle RNA in Regenerated Myeloid Cells after X Irradiation in C3H/He Inbred Mice. Myeloid leukemia cells were derived from regenerated hematopoietic cells damaged by sublethal doses of X radiation in C3H/He inbred mice. We previously found that within the genome of the myeloid leukemia cells, a retrotransposon, the intracisternal A-particle (IAP) element, is integrated. Levels of IAP RNA, the source of cDNA for the integration, were analyzed quantitatively in C3H mice. Higher levels of IAP transcripts were observed in normal cells, particularly in hematopoietic cells, from C3H/He mice, than in those from C57BL/6J and STS/A mice. In the C3H/He mice, an approximately twofold increase in IAP RNA was found in the regenerated spleen and bone marrow cells at 5 days and from 12 to 90 days after whole-body X irradiation. In addition, an increased level of IAP RNA was observed in all the myeloid leukemia cells derived from C3H/He mice. This suggests that the elevated levels of IAP RNA in the regenerated hematopoietic cells after irradiation contribute to the increase in retrotransposition of IAP found in myeloid leukemia cells from C3H/He mice.

Constitutive overexpression of the c-fos gene in radiation-induced granulocytic leukemia in mice

Among various myeloid leukemias which were induced by X rays in C3H/He mice (Seki et al., Radiat. Res. 127, 146-149, 1991), the three most frequent types were analyzed for myeloperoxidase, c-myc, c-myb, and c-fos mRNAs. It was shown by in situ hybridization that all the component cells were positive for myeloperoxidase mRNA in granulocytic leukemia, whereas only half the cells were positive in myelomonocytic leukemia and none in monocytic leukemia. Granulocytic leukemia was also characterized by a persistently heightened expression of c-fos, while the other two types of leukemia showed negligibly low expression of the c-fos message. By contrast, both c-myc and c-myb were expressed to a similar extent in all three types of leukemia. When fresh granulocytic leukemia cells were transferred to culture in a medium containing 0.5% fetal calf serum, c-fos mRNA was decreased rapidly during incubation. The decay of c-fos mRNA was inhibited by cycloheximide markedly but was not changed significantly by actinomycin D. In the culture containing 10% fetal calf serum, the rate of decay of c-fos mRNA was slowed down significantly. Addition of dibutyryl cyclic AMP rapidly restored the c-fos expression and kept it elevated for at least 2 h in the cultured granulocytic leukemia cells. Phorbol ester (TPA) and calcium ionophore A23187 also caused a rapid but transient c-fos expression. A transient c-fos expression was inducible by TPA in the other two types of leukemia cells and in the granulocytic leukemia cells. The results suggest that the persistent expression of c-fos is distinguished from its transient expression and is characteristic for granulocytic leukemia cells as it is for normal mature granulocytes.

Colony-stimulating factor and the proliferation of X-irradiated myeloid stem cells

Abstract Mouse bone marrow cells irradiated in vitro with X-rays (100R or 200R) were cultured for a week in semi-solid agar containing nutrients, horse serum and various amounts of colony-stimulating factor (CSF), and the number of the colonies was counted microscopically. The result showed that the diminution of proliferative activity of myeloid stem cells (CFU-C) induced by X-rays was partly recoverable by increasing the concentration of CSF, and that the irradiated CFU-C required a higher concentration of CSF than did the control CFU-C to produce colonies in the culture. The increased CSF-requirement was not due to the increased liberation of CSF-inactivator or CSF-antagonist in the culture.

Barbiturates enhance retinoic acid or 1,25-dihydroxyvitamin D3-induced differentiation of leukemia HL-60 cells

In the presence of 1 nM retinoic acid (RA), pentobarbital markedly enhanced differentiation of HL-60 cells to granulocytic cells. In the absence of RA, pentobarbital by itself did not induce cell differentiation. Similarly, pentobarbital enhanced the action of 1,25-dihydroxyvitamin D3 to induce differentiation of HL-60 cells into monocyte/macrophage lineage. The potency of various barbiturates to enhance cell differentiation was closely correlated with their activity to inhibit protein kinase C of HL-60 cells. In contrast to staurosporine, however, barbiturates did not affect the action of differentiation inducers of other types such as dimethyl sulfoxide, dibutyryl cyclic AMP or actinomycin D.

Non-oligomeric nature of porcine testicular 20α-hydroxysteroid dehydrogenase

It is known that the majority of pyridine nucleotide-linked dehydrogenases have oligomeric quaternary structures. However, 20a-hydroxysteroid dehydrogenase which was purified from porcine testes [ 1 ] is peculiar in this respect. It behaved as the 35 000 dalton molecule not only in gel-filtration and ultracentrifugation analyses but also in SDSacrylamide gel electrophoresis [ 1 ] thus it is apparently non-oligomeric. In the study reported below, we have demonstrated that the testicular 20a-hydroxysteroid dehydrogenase is not assembled into a polymeric form even while performing its enzymic function.