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Purpose: The infusion of a bacterial solution into the uterus of rats raises the progesterone (P 4 ) concentration in serum and extends diestrus. To understand the origin of the P 4 , we investigated the change in the P 4 concentration of seven groups of rats for 5 days after the infusion of a bacterial solution., Methods: The rats were divided into 7 treatment groups as follows: OvxBac, AdxBac, Ovx, Adx, LapBac, Lap, and Cont. In OvxBac, rats received both ovariectomy and bacterial inoculation into their uterus. In AdxBac, rats received both adrenalectomy and bacterial inoculation into their uterus. In Ovx, rats received only ovariectomy. In Adx, rats received only adrenalectomy. In LapBac, rats received only bacterial inoculation into their uterus. In Lap, rats received only laparotomy. In Cont, rats did not receive any treatment and acted as controls., Results: The P 4 concentration in all treatment groups was higher than in Cont on day 1 (the day following operation) and day 2. In Lap and LapBac, the P 4 concentration was high on day 1 (>30 ng/mL) and maintained that value until day 2. In Adx and AdxBac, the P 4 concentration was average on day 1 (approximately 25 ng/mL) and increased on day 2 to a value close to that of LapBac and Lap. In OvxBac, although the P 4 concentration increased slightly on day 1 and day 2, it reached 22.5 ± 7.5 ng/mL on day 4. In AdxBac and LapBac, the P 4 concentration on day 4 tended to be high., Conclusion: These results suggest that P 4 is initially secreted from the ovaries and the adrenals in response to the surgical stress of laparotomy, and is later secreted from the adrenals due to the inflammatory reaction of the uterus.

Myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1) is a plant enzyme that hydrolyses glucosinolates, principally to isothiocyanates. Myrosinase was purified to homogeneity in good yield from 8-day-old seedlings of Raphanus sativus (daikon) using a four-step procedure involving chromatographies on anion exchange, hydrophobic Phenyl-Sepharose, gel filtration and concanavalin A-Sepharose. In order to stabilize the enzyme and to avoid excessive peak broadening during chromatography, 30% (v/v) glycerol was added to dialysis and chromatography buffers. The purified enzyme was eluted as a single peak from a gel-filtration sizing column with an apparent molecular mass of 120 kDa. The enzyme was resolved into two subunits with molecular masses of 61 and 62 kDa by SDS/PAGE. Ascorbic acid activated the purified enzyme more than 100-fold. The V(max) and K(m) values for the hydrolysis of allyl glucosinolate (sinigrin) were 2.06 micromol/min per mg of protein and 23 microM in the absence of ascorbate and 280 micromol/min per mg of protein and 250 microM in the presence of 500 microM ascorbate, respectively. As the ascorbate concentration was increased from 50 to 500 microM, the V(max) and K(m) values increased in parallel, and thus the V(max)/K(m) ratio remained constant. Similarly, raising the concentrations of sinigrin increased the concentration of ascorbic acid required for half-maximal activation (K(a)). At a sinigrin concentration of 250 microM, the K(a) for ascorbic acid was 55 microM. Sulphate, a reaction product, was a competitive inhibitor of activity, having a K(i) of 60 mM with respect to sinigrin and of 27 mM with respect to ascorbate. Thus activation of myrosinase from R. sativus by ascorbic acid exemplifies an unusual and possibly unique example of linear 'uncompetitive activation' (i.e. a proportionate increase in V(max) and K(m)) of an enzyme. The enzyme also had beta-glucosidase activity and hydrolysed p-nitrophenyl-beta-d-glucopyranoside.

A single subcutaneous injection of a preparation of heat-killed Lactobacillus casei (LC 9018), given before or after irradiation, significantly increased the survival rate of mice that had received 8.5-Gy 137Cs whole-body gamma-irradiation. A similar radioprotective effect was observed when LC 9018 was administered within the period from 2 days before irradiation to 9 h after irradiation, the pre-irradiation treatment being slightly better than the post-irradiation treatment. Increases in the weight of the spleen and in the number of endogenous spleen colonies on days 8 and 12 after irradiation suggested that the radioprotective effect was based on enhanced recovery of hematopoietic tissues. The activity of macrophage colony-stimulating factor (M-CSF) in serum was rapidly increased by the treatment and was maintained at the elevated level for 13 days. At the same time, an increased level of M-CSF mRNA was detected in the livers of the treated mice. However, LC 9018 failed to save the lives of mice when administered 3 days after irradiation, although it increased serum M-CSF as effectively as noted above. The small advantage of the pre-irradiation over the post-irradiation treatment was not explained by the increases of metallothionein in the hematopoietic tissues of the treated 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.

X-irradiation-induced emesis was investigated in Suncus murinus, a house musk shrew. Whole body X-irradiation caused emesis, and the calculated ED50 value that induced emesis in 50% of animals was 429 cGy. At the irradiation dose of 800 cGy all the animals vomited 10.0 +/- 2.4 times with a latency of 20.0 +/- 2.9 min. The emetogenic effect of X-irradiation was dependent on the part of the body exposed. Abdominal X-irradiation at 1000 cGy caused emesis in all animals studied, whereas the same dose to the head had no emetogenic effect. We investigated several prophylactic methods against X-irradiation-induced emesis. Surgical vagotomy completely inhibited the emesis induced by 800 cGy X-irradiation. Emesis was also prevented by the subcutaneous administration of tropisetron (ICS 205-930, a selective serotonergic 5-HT3 receptor antagonist) with an ID50 value of 29 micrograms/kg. These results suggest that (1) suncus is a useful experimental animal for the study of radiation-induced emesis and the development of prophylactic drugs, (2) serotonin plays an important role in X-irradiation-induced emesis, and (3) X-irradiation-induced emesis is very similar to that caused by cancer chemotherapeutic agents.

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.

The activity of macrophage colony-stimulating factor (M-CSF) was found in the culture supernatant of mouse parenchymal liver cell fractions in a bone marrow colony-forming assay. The activity of an M-CSF-like substance purified by a four-step procedure was neutralized by goat anti-mouse M-CSF antiserum. M-CSF mRNA was detected in cellular RNA prepared from cultured parenchymal liver cell fractions by Northern blot analysis and also in cultured parenchymal liver cells by in situ hybridization. These results indicate that parenchymal liver cells have the capacity to produce M-CSF. We discuss the role of M-CSF in hematopoiesis, the immune response, and other biological phenomena.

Serum thymic factor (FTS) reduced mortality of mice after total-body irradiation with 7.56 Gy X rays. The radioprotective effect was achieved by daily repeated subcutaneous injections of 3-100 micrograms FTS, while doses higher than 300 micrograms/day/mouse were neither radioprotective nor toxic. Similarly, degeneration of the spleen was moderated by 3-100 micrograms FTS but not by 500 micrograms FTS in sublethally (3.78 Gy) irradiated mice. Histological examination showed that hematopoiesis was enhanced in the spleen by daily injections of 10 micrograms FTS. Spleen cells from the FTS-treated mice incorporated more [3H]thymidine in culture with or without concanavalin A. The treatment with FTS increased the production of colony-stimulating factor in the spleen as well as in peritoneal macrophage-like cells, and caused a significant increase in the number of granulocyte-macrophage colony-forming cells both in the spleen and in the femoral bone marrow. Furthermore, FTS prevented a decrease in circulating neutrophils in the sublethally irradiated mice. Prominent overshoot recovery of myelopoiesis, which occurred occasionally in sublethally irradiated mice, did not occur in the FTS-treated mice. The decrease in blood erythrocytes was also significantly reduced. These observations imply that this thymic hormone has potential as a radioprotector.

Long-term administration of (-)-epigallocatechin 3-O-gallate (EGCG) to mice through drinking water prevented radiation-induced increase of lipid peroxides in liver and significantly prolonged life span after lethal whole-body X-irradiation. The result indicates validity of this green-tea component as an orally active radio-protector of very low toxicity.

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.

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.

A rapid, simple, and efficient method for DNA blotting is presented. This method is characteristic in that DNA is transferred without denaturation from an electrophoretic gel to a membrane in low-salt concentrations and denatured on the membrane after blotting. More than 89% of double-stranded DNAs ranging in size from 75 to 1.9 million bp can simultaneously be transferred from the gel to a positively charged nylon membrane within 10 min. The present "low-salt electroblotting" method is superior to other blotting methods in that it saves time and labor and its high and even transfer efficiency makes it useful for hybridization analysis.

Dialyzed urine of mice bearing leukocytosis-inducing fibrosarcoma stimulated granulocyte colony formation in semisolid agar culture of human bone marrow cells. Removal of phagocytic cells prior to stimulation did not interfere with the formation of these colonies in the culture. On the other hand, macrophage colonies were predominantly produced when murine bone marrow cells were stimulated by the dialyzed mouse urine. The activity of colony-stimulating factor (CSF) in the urine of normal mice was less than 1/100 of that in the urine of tumor-bearing mice. DEAE-cellulose column chromatography separated the activity stimulating human granulocyte colony formation from that stimulating murine macrophage colony formation. Further purification showed that a sialoglycoprotein with an apparent molecular weight of 80,000 corresponded to the macrophage CSF, which was devoid of activity toward human cells. The molecular properties of the human-active granulocyte CSF could not be studied further, because it was quite unstable.

Among various immunomodulating agents examined, OK-432 (Streptococcus pyogenes cell bodies), BCG (bacillus Calmette-Guerin) and LPS (lipopolysaccharide) were effective in enhancing the production of granulocyte/macrophage colony-stimulating factor (GM-CSF) in the cultures of mouse spleen cells. Control non-stimulated spleen cell cultures accumulated GM-CSF activity maximally on day 3 and lost it subsequently within a few days. On the other hand, a large amount of GM-CSF activity could be detected in the medium until day 9 when OK-432 was added in the culture at the start of incubation. The GM-CSF obtained from OK-432 or LPS stimulated cultures and the GM-CSF obtained from the control non-stimulated culture eluted from an Ultro-gel AcA 34 column at a similar position and inactivated similarly at 80 degrees C. However, these three GM-CSFs behaved in slightly different manners upon isoelectrofocusing. These results suggest that the GM-CSFs produced in increasing amounts by the stimulated spleen cells are similar to that produced by the control non-stimulated cells, but slightly different from one another in the extent of sialylation according to the difference of the stimulant.

A clone, YS-T22, of cells from Yoshida sarcoma cell line, YSSF-212T, grown in "serum-free" culture medium produced factors stimulating differentiation of mouse myeloid leukemia cells (M1) to macrophages and granulocytes. The formation of macrophages and granulocytes was accompanied by induction of phagocytosis, locomotive activity, and lysosomal enzyme activities. The rates of induction of these differentiated phenotypes were proportional to the concentration of the factor added and the period of treatment. The factor stimulating differentiation of M1 cells was a heat-labile, nondialyzable proteinaceous substance that was inactivated by trypsin but not by ribonuclease or glycosidases. On diethylaminoethyl cellulose chromatography, the factor stimulating differentiation of M1 cells from conditioned medium of YS-T22 cells was eluted in various fractions with or without activity of the colony-stimulating factor.

A macrophage colony-stimulating factor (M-CSF) was purified to homogeneity from a large amount of normal human urine. Microanalysis of the N-terminal amino acid sequence up to residue 44 revealed only a single residue difference from that deduced by other workers from the nucleotide sequence of M-CSF cDNA clones. The amino acid composition of the present preparation suggested that the M-CSF which we purified possessed a structure fitting the sequence 1-190 of TPA30-1 cell M-CSF deduced by Wong et al. [(1987) Science 235, 1504-1508].