Human myelogenous leukaemic cells can be induced to differentiate into the monocyte/macrophage pathway by protein inducers called differentiation inducing factors (DIF) in conditioned media of mitogen-stimulated human peripheral blood leukocytes. However, human DIF has not yet been well characterized. DIF is known to be a T-cell lymphokine, as it can be obtained from the T-cell line HUT-102 and can be partially purified from medium conditioned by phytohaemagglutinin (PHA)-stimulated lymphocytes. We found that monocytes also produce factor(s) that induce differentiation of human myelogenous leukaemia cell lines to cells with macrophage-like characteristics. This factor(s) has activity different from that of colony-stimulating factor(s) or interferons. We have now purified a DIF to homogeneity from medium conditioned by PHA-stimulated leukocytes using a human myeloblastic leukemia cell line, ML-1, as target cells. The purified DIF has a relative molecular mass (Mr) of approximately 17,000, with an NH2-terminal sequence the same as that of human tumour necrosis factor (TNF). Recombinant human TNF (rHuTNF) induces differentiation of ML-1 cells and an anti-pDIF monoclonal antibody can neutralize both differentiation inducing activity and cytotoxic activity of DIF and rHuTNF. The findings indicate that one of the DIF(s) produced by leukocytes is probably TNF.
Fatty aldehydes, present in the luminescent cells of Photobacterium phosphoreum and Achromobacterfischeri, and td a very slight extent in the cells of a visually dark, "aldehydeless" mutant of the latter species, were extracted, purified, and oxidized to the corresponding acids. The acids were analyzed by mass spectrometry. The results, in Cell-free extracts of lumitous bacteria that produced an NADH-activated luminescence were reported in 1953 (1). Evidence soon followed that the system required FMN (2) and, in addition, a factor "KCF" obtained from kidney cortex (3), subsequently identified as hexadecanal (4). This aldehyde could be replaced by any one in a series of saturated, straightchain aldehydes having chain lengths from C7 to C16 with varying degrees of effectiveness (5). Until now, however, no evidence has been reported that such aldehydes are specifically required for luminescence of the bacterial system in vivo. In fact, the specific requirement of an aldehyde is questionable on the grounds that a terminal nitrite group can satisfactorily replace the terminal aldehyde group (6).Recent evidence has shown that the above type of aldehydes of chain lengths from C9 to C14 are oxidized to the corresponding acids by the bacterial luminescent system in vitro (7-9), with a quantum yield for each aldehyde amounting to 0.17 0.01 photons per aldehyde molecule reacted (7). Suggestive evidence that aldehydes function in luminescence also in vivo has been available in the fact that certain dark mutants, which seem identical to the parent strain except for failing to emit visible luminescence, respond practically at once by emitting a bright glow of light when a suitable aldehyde is added to the dark cells (10). Moreover, an aldehydogenic compotond in extracts of Photobacterium phosphoreum has been described (11).The present study demonstrates the presence of saturated fatty aldehydes in luminescent bacterial cells of Achromo- During extraction and purification of aldehydes from the bacteria, assays were made, to monitor the procedure at each step, on the basis of the amount of light emitted with a bacterial luciferase preparation from A. fischeri. To a specimen containing aldehyde for assay, a combination of the luciferase preparation, FMN, and NADH was added, and the total luminescence was measured under conditions described (see explanation of Fig. 2, p. 2088, ref. 7).Preparation of Bacterial Cells. Each species Was cultivated on the surface of hard agar, consisting of 30 g of Difco BactoNutrient Agar Dehydrated per liter of 3% (w/v) NaCl, plus 10 ml of glycerol, plus 5 g of powdered CaCO3, autoclaved, and aseptically poured into sterile aluminum baking pans with sheet aluminum covers, or into sterile fiberglass-type pans with covers, to a depth of about 0.5 inch (1.3 cm) (the deeper than usual medium favors better growth and luminescence).A total surface area of 1.3 m2 -was inoculated for each extraction. The growth of A. fischeri was harvested after incubation for 24 hr at 250, and that of P. phosp...
Tubuloreticular inclusions (TRI) were induced in normal blood lymphocytes after incubation with Staphylococcus aureus Cowan 1 (STA), but they were not induced by pokeweed mitogen (PWM), as we reported previously. TRI were also induced in Raji cells when grown in the medium of STA culture. Alpha-interferon (alpha IFN) was detected only in the medium of STA culture and not in PWM culture. The cells of PWM cultures formed TRI when exposed to various concentrations of human leukocyte alpha IFN. The incidences of TRI-positive cells in the presence of 50-500 IU/ml of alpha IFN were 3-5% on day 2 and increased to 10% on day 7. On days 5-7 of the PWM cultures, plasmacytoid cells containing TRI were seen not infrequently. In the presence of a high concentration of alpha IFN (10,000 IU/ml), which was sufficient to inhibit cell growth and differentiation, the growth of the TRI region was not altered and the incidence of TRI-positive cells was 9% on day 2 and increased to 15% on day 7. Our observations suggest that the TRI formation in STA culture is attributable to the alpha IFN produced endogenously by STA-stimulated cells and that some relationship might exist between the incidences of TRI-positive cells in these mitogen-stimulated cultures and the biologic functions of IFN.
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