Normal mouse lung tissue produces a growth-inhibitory factor(s) preferential for mouse monocytic leukemia cells

Summary A growth-inhibitory (GI) factor, that specifically inhibits the growth of mouse monocytic leukemia cells, was found in conditioned medium of mouse lung tissue, but not in that of mouse brain, heart, liver, or kidney tissue. Conditioned medium of spleen or bone marrow cells had low GI activity. Pulmonary macrophages were as active as peritoneal and bone-marrow-derived macrophages in production of the GI activity. The GI factor inhibited the growth of murine monocytic leukemia cell lines Mm-A and J774.1, but scarcely inhibited the growth of other mouse cell lines, such as a myeloblastic leukemia cell line (M1), a Friend erythroleukemia cell line (745A) and a mammary carcinoma cell line (FM3A). It had no significant effect on the growth of human monocytic leukemia cell lines U937 and THP-1 or on the HL-60 promyelocytic leukemia cell line. These results suggest that the GI factor produced by mouse lung tissue preferentially inhibits the growth of mouse monocytic cells.The GI factor was found to be a proteinaceous substance with a molecular mass of 25 kDa. On chromatofocusing, the GI activity was eluted with Polybuffer 96/acetic acid at pH 7.2–7.5. The GI activity was not significantly decreased by heat treatment at 56°C for 30 min or acid treatment (0.01 M HCl, 14 h), but the GI activity in glycosidase-treated conditioned medium of lung tissue was lost on heat treatment. The GI activity could not be neutralized with anti-(interferon + ) antibody. The activity was produced constitutively by lung tissues and its production was not stimulated appreciably by lipopolysaccharide, lectin, or poly(I)·poly(C). The GI factor appears to be a cytokine unrelated to known cytokines such as tumor necrosis factor, interleukin-1, transforming growth factor , and interferons. These results suggest that the GI factor may be involved in negative feedback regulation of macrophage production in steady-state conditions in the lungs.This work was supported in part by a grant for Cancer Research from the Ministry of Education, Science and Culture, and a grant from the Ministry of Health and Welfare for a Comprehensive 10-Year Strategy for Cancer Control     

Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells

A procedure is described for purification of a factor (D-factor)-inducing differentiation of mouse myeloid leukemic cells (M1) into macrophages from serum-free mouse L929 cell-conditioned medium. The procedure included ammonium sulfate precipitation, DEAE-cellulose, Sephadex G-200 and phenyl-Sepharose column chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel-filtration column. The purified factor gave a single band of protein with a molecular weight of 62,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis which coincided with biological activity. Its half-maximal concentration for inducing differentiation of M1 cells into macrophages was 1.7 X 10(-11) M. Even at 2.6 X 10(-9) M, it did not induce colony formation of normal bone marrow cells, suggesting that it was distinct from the growth factor for normal precursors of macrophages and/or granulocytes.     

Partial purification and initial characterization of a novel differentiation factor for mouse myeloid leukemia cells

We found cells spontaneously differentiated from mouse myeloid leukemia M1 cells were producing a strong differentiation factor in culture medium and established a method to prepare a large quantity of conditioned medium containing the differentiation factor. The factor purified over 4,000-fold from the conditioned medium showed a single peak due to a peptide on a TSK 3000PW column which was coincident with differentiation activity. The molecular weight of the factor estimated by high-performance gel filtration chromatography was 1,300, which is remarkably lower than the values reported for protein differentiation factors reported thus far. M1 cells were induced to differentiate into macrophage-like cells by the factor.     

Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor

A naturally occurring inducer of terminal differentiation in a murine myelomonocytic leukemia cell line (WEHI-3B) was purified to apparent homogeneity from medium conditioned by lungs from mice injected with bacterial endotoxin. The factor was purified over 400,000-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Bio-Gel P-60 in 1 M acetic acid, reverse-phase high performance liquid chromatography on a phenyl-silica column, and high performance liquid chromatography on a gel filtration column. During the first two steps, the differentiation-inducing factor was separated completely from a known proliferative regulator for normal myeloid cells, granulocyte-macrophage colony-stimulating factor, but it co-purified through all remaining steps with a distinct granulocyte-specific colony-stimulating factor. The purified factor showed a single protein band of Mr = 24,000-25,000 on sodium dodecyl sulfate-polyacrylamide gels coincident with both differentiation-inducing and granulocyte colony-stimulating activity. The granulocyte-specific colony-stimulating factor was active on WEHI-3B cells and normal granulocytic progenitor cells in vitro at the same half-maximally active concentration of 3 X 10(-12) M.     

Purification of a murine leukemia inhibitory factor from Krebs ascites cells

A factor capable of inducing terminal differentiation in the murine myeloid leukemia cell line M1 has been purified to apparent homogeneity from the medium conditioned by Krebs II ascites tumor cells. The factor, termed leukemia inhibitory factor (LIF) is a single chain glycoprotein of apparent Mr 58,000 which induces differentiation and inhibits proliferation of the M1 cell line but not the WEHI-3B D+ murine myeloid leukemic cell line and has no detectable proliferative activity on normal myeloid progenitor cells. It was purified using four successive high-efficiency purification steps--anion-exchange chromatography on DEAE-Sepharose; cation-exchange chromatography on CM-Sepharose; affinity chromatography on lentil lectin-Sepharose; and reverse-phase high-performance liquid chromatography on a phenyl-silica matrix--to a specific biological activity of approximately 1.25 X 10(8) units/mg with an overall purification of 12,000-fold and a yield of 73% for the activity failing to bind to DEAE-Sepharose. Sufficient quantities of the factor (12 micrograms, 200 pmol) have been purified to allow structural and functional analysis of the molecule and comparison with other know differentiation inducers.     

Purification of a multipotential colony-stimulating factor from pokeweed mitogen-stimulated mouse spleen cell conditioned medium

A factor able to stimulate the proliferation and differentiation of multipotential stem cells and progenitor cells of the granulocyte-macrophage, eosinophil, and erythroid lineages as well as being able to maintain factor-dependent cell lines in culture has been purified from pokeweed mitogen-stimulated mouse spleen cell-conditioned medium. The factor was purified over 2 million-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-Sepharose, reverse-phase high performance liquid chromatography on a phenyl-silica column, and gel permeation high performance liquid chromatography. All of the biological activities ascribed to the multipotential colony-stimulating factor co-fractionated through all steps, and the other known mouse-active hemopoietic regulator in pokeweed mitogen-stimulated mouse spleen cell-conditioned medium, granulocyte-macrophage colony-stimulating factor, was separated at the ion exchange step. Two protein species having Mr = 24,000 and 19,000 were visualized by silver-staining of sodium dodecyl sulfate-polyacrylamide gels of the purified factor. Both species migrated coincidently with the biological activities. The factor was active at a half-maximal concentration of 1 X 10(-13) M when assayed on a factor-dependent cell line.     

Purification and characterization of a growth factor from guinea pig harderian gland

A polypeptide growth factor, Harderian gland-derived growth factor (HGDGF), has been purified approximately 43,000-fold from guinea pig Harderian gland by column chromatography on TSK gel DEAE-5PW, blue-Sepharose CL-6B, and Superose 12. The yield was approximately 10%. The Superose 12 fraction was further purified by Aquapore BU-300 reversed-phase chromatography to apparent homogeneity. HGDGF was eluted from TSK gel DEAE-5PW at 0.20-0.35 M NaCl, with a linear gradient of 0.15-0.80 M NaCl and at 2.20 M NaCl from blue-Sepharose CL-6B. The activity of HGDGF toward human embryonic cells (TIG-3) was quantitated, [3H]thymidine incorporation for 48 h being stimulated in a linear and dose-dependent manner. Purified HGDGF has a molecular weight of approximately 13,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve column chromatography. HGDGF is labile to treatment with SH reagents or acetic acid. Both trypsin digestion and boiling decrease the activity of HGDGF. Its pI is 5.1. HGDGF stimulates the multiplication of TIG-3 cells but has no effect on human endothelial cells K2T1 or A2T2 which require fibroblast growth factor for growth. HGDGF appears to differ from other growth factors, suggesting that it is a previously undescribed growth factor.     

Mitogenic polypeptide of the mammalian seminiferous epithelium: biochemical characterization and partial purification

A mitogenic polypeptide, previously identified in Sertoli cells of the prepuberal mouse (Feig, L. A., A. R. Bellve, N. Horbach-Erickson, and M. Klagsbrun, 1980, Proc. Natl. Acad. Sci. USA., 77:4774-4778), now has been shown to exist in Sertoli cells of the adult mouse and in the seminiferous epithelium of several other mammalian species, including the rat, guinea pig, and calf. The levels of this seminiferous growth factor (SGF) are not appreciably reduced in adult mouse testes following hypophysectomy. SGF purified from either the adult mouse or newborn calf seminiferous epithelium has a molecular weight (Mr) of 15,700 and a pl between 4.8 and 5.8, when exposed to denaturing conditions. Furthermore, SGF from these two mammalian species probably has few exposed hydrophobic domains and has a strong propensity to aggregate into multiple, high Mr species. A purification sequence based on these biochemical properties has enabled a greater than 350-fold enrichment of SGF activity from the calf seminiferous epithelium. The protocol involves a sequence of: (a) ammonium sulfate precipitation, (b) DEAE-cellulose ion exchange chromatography, (c) gel filtration chromatography on Bio-Gel P150 in 1.0 M ammonium acetate, (d) hydrophobic chromatography on dodecyl agarose, and (e) gel filtration chromatography in 6.0 M guanidine hydrochloride. Subsequent analysis of this purified preparation by SDS PAGE, followed by silver staining, reveals approximately 7 polypeptides with Mr between 14,000 and 20,000.     

Selection of mouse macrophage-like sublines that differ in leukemogenic potential and characterization

The murine macrophage-like cell line (Mm-1), which is nonleukemogenic to syngeneic SL mice, was originally derived from spontaneously differentiated cells of a clonal line of mouse myeloid leukemia cells (M1). In the present experiment, variant cell lines with a high (Mm-A), moderate (Mm-P), and little or no (Mm-S1 and Mm-S2) leukemogenic potential were obtained from the Mm-1 cells. The mean survival times of syngeneic SL mice inoculated i.p. with 5 X 10(6) Mm-A and Mm-P cells were 17 and 33 days, respectively, whereas almost all the mice inoculated with Mm-S1 or Mm-S2 cells survived for more than 90 days. These variant cell lines did not lose their macrophage-like characteristics in vitro. These variant cell lines phagocytized latex beads and sensitized sheep erythrocytes, produced lysozyme, and adhered to culture dishes. The four variant cell lines showed no significant difference in proliferation rates in vitro in liquid medium containing 10% calf serum, but Mm-A cells could grow both in soft agar medium in the absence of ascitic fluid containing colony-stimulating factor (CSF) and in liquid medium containing 1% serum, whereas Mm-P cells could grow in the liquid medium but not in soft agar medium without ascitic fluid, and Mm-S1 and Mm-S2 cells could not grow in either medium. The ratio of the nuclear area to the cell area (NCR) of Mm-A cells was a high (51%) but those of Mm-S1 and Mm-S2 cells were low (40-41%), and that of Mm-P cells was intermediate (44%). The leukemogenicity of Mm-1 cell lines was roughly correlated with their NCR. The possibility that interactions between Mm-1 variant cells and host immune cells might be involved in the mechanisms of their different leukemogenicities was not supported by results on the in vitro susceptibilities of Mm-1 variant cells to the cytostatic actions by normal macrophages and spleen cells and on leukemogenicities of the Mm-1 variant cells in athymic nude mice. A possible method of control of the leukemogenicity of Mm-1 variant cells is discussed.     

Purification of a monocyte chemotactic factor secreted by nonhuman primate vascular cells in culture

A protein chemotactic for peripheral blood monocytes (SMC-CF) of potential importance in their recruitment to the arterial intima in atherogenesis was purified from serum-free medium conditioned by cultured baboon aortic medial smooth muscle cells. The purification of SMC-CF was monitored by a filter assay using human peripheral blood mononuclear cells and was achieved by batch separation on a cation-exchange gel followed by gel permeation chromatography, ion-exchange high-performance liquid chromatography (HPLC), and reversed-phase HPLC. The overall recovery was approximately 10% of the initial activity and yielded 0.5-1 microgram of SMC-CF/L of conditioned medium. On analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SMC-CF migrated as a monomeric protein with an apparent molecular weight of 14,500. A dose-dependent relationship was observed between SMC-CF concentration and monocyte chemotactic activity, with maximal and half-maximal biologic activity being observed at approximately 5 and 0.1 nM, respectively. Cultured baboon aortic smooth muscle cells also express the genes for both the A and B polypeptide chains of platelet-derived growth factor, which has been reported to be chemotactic for blood monocytes and neutrophils [Deuel, T. F., Senior, R. M., Huang, J. S., & Griffin, G. L. (1982) J. Clin. Invest. 69, 1046-1049]. Amino acid composition analyses indicate that SMC-CF is not derived either from polypeptide chain of this growth factor or from certain potentially chemotactic connective tissue proteins.     

Purification of guinea pig tumor necrosis factor (TNF): comparison of its antiproliferative and differentiative activities for myeloid leukemic cell lines with those of recombinant human TNF

Recently, we suggested that the effect of differentiation inducing factor (D-factor) which is found in the supernatant of macrophages, and induced the differentiation of a mouse myeloid leukemic cell line, M1, into macrophage-like cells, may be a result of the cooperative effects of tumor necrosis factor (TNF) and interleukin 1 (IL-1). In this study, we purified guinea pig (G.P.) TNF secreted from peritoneal macrophages and compared the antiproliferative and differentiative effects of the G.P. TNF with those of recombinant human TNF (rHuTNF). The purification scheme consisted of ultrafiltration, gel filtration-high performance liquid chromatography (HPLC), DEAE-HPLC, and reverse-phase HPLC. The cytotoxic activity of the purified substance was approximately 1.5 x 10(8) U/mg. The isoelectric point was 5.2. The molecular weight was 40 to 45 kDa as estimated by gel filtration and 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal amino acid sequence was determined to be Ser-Ala-Ser-Gln-Asn-Asp . . . . Approximately 76 or 71% homology between G.P. TNF and mouse or human TNF exists in the NH2-terminal 21 residues. The purified G.P. TNF and rHuTNF demonstrated D-factor activity only in the presence of recombinant human IL-1 alpha in M1 cells. We also determined the effect of TNF on two human myeloid leukemic cell lines (THP-1 and U937). The purified G.P. TNF and rHuTNF inhibited the growth of U937 cells, but did not induce their differentiation. In THP-1 cells, TNF slightly inhibited the growth and induced differentiation. In mouse cell lines G.P. TNF was more effective than rHuTNF for differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of a mammary-uterine-pituitary tumor cell growth factor from pregnant sheep uterus

A mammary-uterine-pituitary tumor cell growth factor has been purified from lyophilized powders of pregnant sheep uteri by a five-step procedure. Uterine-derived growth factor (UDGF) was extracted from the powders with 0.1 M acetic acid, heated at 95 degrees C, and further purified by sulfopropyl-Sephadex C-25, Sephadex G-50, and carboxymethyl-Sephadex C-25 chromatography. From 500 g of uterine powder, 40 to 50 mg of UDGF can be isolated at an overall yield of 33%. The degree of homogeneity of the final preparations was estimated by 8 M urea, 0.1% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), and by PAGE under nondissociating conditions at either pH 8.5 or 4.5. In all PAGE experiments, the purified UDGF preparation showed a single Coomassie blue-stained band that directly corresponded to the only area of elution of UDGF activity from duplicate unstained gels. Molecular sieve high performance liquid chromatography HPLC, reverse phase HPLC on an octylsilyl (C8) column, and hydrophobic chromatography on octyl-Sepharose CL-4B all confirm a similar degree (i.e. greater than 90%) of homogeneity. The Mr of UDGF estimated by urea/sodium dodecyl sulfate-PAGE was 4200 +/- 500 and, by molecular sieve HPLC, 6200 +/- 1000. The isoelectric point of UDGF was estimated as pI = 7.3. The UDGF isolated showed marked cell-type specificity for established cell lines that were derived from estrogen-responsive tumors; purified sheep UDGF was mitogenic for MTW9/PL rat mammary tumor cells (at 10(-10) to 10(-9) M concentrations) while showing no mitogenic activity toward normal rat diploid fibroblasts. UDGF also promoted growth of uterine-derived tumor cells and the GH3/C14 rat pituitary line. Measuring growth as an increase in cell number, UDGF supported the logarithmic growth of the MTW9/PL rat mammary tumor cells over 6 days; other known hormones and growth factors were not able to substitute for the UDGF mitogenic action on MTW9/PL cells. It is concluded that a rapid, high-yield method of purification of a new uterine-derived growth factor activity has been developed.     

A transforming growth factor-beta like growth factor in mouse embryonal carcinoma cells

Our previous study indicated that polypeptides isolated from acid/ethanol extracts of solid tumors of a cloned F9-3 embryonal carcinoma cells by Bio-Gel P60 column chromatography were found to be able to stimulate anchorage independent growth of either NIH 3T3 cells or NRK 49 F cells in soft agar. The major peak of active elute had a molecular weight of about 15 kDa as determined by SDS-polyacrylamide gel electrophoresis. In the present report we further isolated and purified the active compound corresponding to molecular weight of 15 kDa by gel filteration on Bio-Gel P10 column (Fig. 1) and then by high pressure liquid chromatography (Fig. 2). It was found that the purified 15 kDa molecules showed some properties similar to transforming growth factor-beta (TGF-beta): 1. Colony-stimulating activity in soft agar can be induced in NRK 49 F cells only in the presence of mouse epidermal growth factor (EGF) (Plate I); 2. Increase in relative uptake of 3H-thymidine in NRK 49 F cells occurred in the presence of EGF, but with the same amount of EGF, not much change in 3H-thymidine incorporation could be found with further increasing amounts of purified 15 kDa molecules (Fig. 3); 3. Like human blood platelets derived TGF-beta, inhibition effect on the growth of mink lung epithelial cells (CCL/64) can also be exhibited by purified 15 kDa molecules (Fig. 4). In addition, using ELISA procedure, we have also demonstrated that the 15 kDa molecules had immunological reactivity with the antibody raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta 1 from human blood platelets (Fig.5). Thus, the 15 kDa molecules isolated from mouse F9-3 embryonal carcinoma cells appeared to share some common antigenic determinants with human TGF-beta 1 molecule. These results taken together provide strong support for the existence of TGF-beta like growth factor in mouse embryonal carcinoma cells.     

Purification and characterisation of a growth factor from porcine bone

A growth factor was extracted from porcine bone matrix by demineralisation and purified by heat and acid treatment, hydroxyapatite chromatography and gel filtration under dissociative conditions and reverse-phase HPLC. Using the mitogenic response of osteoblast-progenitor cells from embryonic chicken, a mitogenic activity was purified 3000-fold. The mitogenic protein thus purified shows an apparent molecular mass of 13.5 kDa in both the nonreduced and reduced form on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The mitogenic activity is sensitive to proteinase K, dithiothreitol, and resistant to DNAse, RNase, heat (70 degrees C) and pH (3-10). The factor stimulates the proliferation of osteoblast-progenitor cells from embryonic chick at a concentration of 1 ng/ml. It is active on cells from skin, periosteum and sternum and has no or little activity on cells of the calvaria, intestine or kidney of embryonic chick or on mouse AKR-2B/Balb c/3T3 cell line.     

Hormonal regulation of insulin-like growth factor I secretion by bovine adrenal cells

The role of insulin-like growth factor I (IGF-I) on the specific function of several steroidogenic cells has been recently reported. Since IGF-I is produced by several tissues, we have investigated whether bovine adrenal cells secrete this peptide. Purification of conditioned medium from adrenal cells incubated with [35S]methionine through affinity chromatography (monoclonal anti-IGF-I antibody), high pressure liquid chromatography, and polyacrylamide gel electrophoresis revealed a single band of similar Mr as pure recombinant IGF-I. Moreover, the purified adrenal-secreted IGF-I displaced bound 125I-IGF-I to its adrenal receptors, and pretreatment of adrenal cells with the purified peptide enhanced the acute corticotropin (ACTH)-induced cAMP production as recombinant IGF-I. The basal secretion of IGF-I (6 +/- 1 ng/48 h/10(6) cells) was stimulated 3-, 4.5-, and 9.5-fold by fibroblast growth factor, angiotensin II (A-II), and ACTH, respectively, but not by growth hormone. The stimulatory effects of A-II and ACTH were dose-dependent (ED50 congruent to 2.5 x 10(-8) and 1.5 x 10(-10) M, respectively), and the effects of both hormones were additive. Glucocorticoids were not the mediators of the effect of the two hormones on IGF-I secretion, since inhibition of their steroidogenic action by aminoglutethimide did not significantly modify IGF-I secretion. An immunoreactive IGF-I material was also secreted by mouse adrenal tumor cell line Y-1, but the stimulatory effect of ACTH was only 2-fold, and there was no effect of A-II. Since bovine adrenal cells contain specific IGF-I receptors and this peptide is required for the maintenance of some adrenal cell-specific function, the present data suggest that IGF-I may act in an autocrine fashion to stimulate adrenal cell differentiation stimulated by ACTH and A-II.     

雷公藤植物内生Micromonospora sp. M66生产的一组吲哚生物碱

【目的】研究稀有放线菌——雷公藤内生小单孢菌(Micromonospora sp.M66)的次级代谢产物,为微生物药物或农用生物制剂开发提供结构多样的化合物资源。【方法】利用薄层层析、正(反)相硅胶柱层析、凝胶层析、液相色谱等技术对M66菌株中次级代谢产物进行分离纯化,利用波谱技术对化合物进行结构鉴定。【结果】最终分离纯化了7个单体化合物,结合质谱与核磁技术对这7个化合物进行了结构解析和鉴定,它们属于一组吲哚生物碱。化合物2是重要的植物生长调节剂,化合物3对淋巴细胞性白血病细胞P388、枯草芽孢杆菌和酿酒酵母的增殖有抑制作用,化合物6对金黄色葡萄球菌有很好的抑制作用。【结论】化合物3-7首次从小单胞菌中鉴定出来,表明该小单孢菌具有较强的利用吲哚或色氨酸合成次级代谢产物的能力和挖掘生物碱类药物的潜力。     

Anchorage-dependent growth factor(s) produced by rat sarcoma (XC) cells.

The autocrine growth factor(s) was isolated from serumfree conditioned medium of rat sarcoma (XC) cells. Autocrine activity was enriched by ultrafiltration using Amicon YM 10 membrane, extraction with 1 M acetic acid and partially purified (650-fold) by chromatography on Bio-Gel P-100 and P-60. The final recovery of the autocrine factor(s) was 4 micrograms from 1800 ml of the conditioned medium (a yield of 6%). The factor(s) with molecular weight 6-10 kDa was heat and acid stable but inactivated by trypsin and dithiothreitol. It stimulated anchorage-dependent (but not anchorage-independent) growth of XC cells as well as untransformed, established lines of rat (NRK) and mouse (3T3) cells. The results obtained may suggest that autocrine factor(s) produced by XC cells can be one of EGF-like or/and insulin-like growth factors.     

Diacyltrehalose of Mycobacterium tuberculosis inhibits lipopolysaccharide- and mycobacteria-induced proinflammatory cytokine production in human monocytic cells

The lipids located in the outer layer of Mycobacterium tuberculosis, which include sulfolipid, phthiocerol dimycocerosate (PDIM), diacyltrehalose, and polyacyltrehalose, may play a role in host-pathogen interactions. These lipids were purified using thin-layer chromatography, and their ability to induce proinflammatory cytokines in human monocytes and in a human acute monocytic leukemia cell line (THP-1) was examined. None of the lipids tested induced significant interleukin (IL)-12p40 or tumor necrosis factor (TNF)-alpha production in monocytic cells. Diacyltrehalose significantly inhibited lipopolysaccharide- and M. tuberculosis-induced IL-12p40, TNF-alpha, and IL-6 productions in human monocytes, whereas other lipids had no effect. However, diacyltrehalose was unable to inhibit peptidoglycan-induced IL-12p40 production. These results suggest that diacyltrehalose is a mycobacterial factor capable of modulating host immune responses.     

Purification of a factor inhibiting differentiation from conditioned medium of nondifferentiating mouse myeloid leukemia cells

Mouse myeloid leukemic M1 cells are induced to differentiate by various differentiation inducers. Activity for inhibition of induction of differentiation of M1 cells (I-factor activity) was detected in conditioned medium of variant M1 cell clones that were resistant to differentiation inducers, and this I-factor activity was shown to be closely associated with resistance of the cells to differentiation inducers. In this work, the I-factor was purified to apparent homogeneity from conditioned medium of resistant M1 cells. The purification procedure consisted of ammonium sulfate precipitation, CM-Sepharose CL-6B, Sephadex G-200, reverse-phase high performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The factor was analyzed by radioiodination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The purified factor gave a single band of protein with a molecular weight of 68,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis which coincided with its biological activity. The concentration of I-factor required for 50% inhibition of dexamethasone-induced differentiation of M1 cells was 24 pM. At its effective concentration it had no effect on cell proliferation, and even at 1.2 nM it did not inhibit colony formation of normal bone marrow cells, suggesting that it was distinct from the inhibitor of normal precursors of macrophages and/or granulocytes.     

Toxic substance from a natural bloom of Microcystis aeruginosa.

A toxic substance contained in the blue-green alga Microcystis aeruginosa was purified and partially characterized. Toxic algal cells were collected from a highly eutrophic lake in Japan, and the toxin was purified by homogenization, ultrafiltration, gel filtration, and ion-exchange chromatography. The final preparation gave a single peak on high-performance liquid chromatography. The toxicity was somewhat less than that reported for other toxins from this alga. The water extract of 6.7 mg (dry weight) of cells and 72 microgram of the purified protein was required to kill a mouse (1 mouse unit). The main amino acids of the toxin were glutamic acid, asparatic acid, alanine, glycine, arginine, and leucine. The molecular weight of the toxin was 2,950 as determined by high-performance liquid chromatography.