Susceptibility of autologous target cells to lysis by lymphokine-activated effectors from interferon-α-treated chronic myelogenous leukaemia patients

Summary Chronic myelogenous leukemia (CML) patients in chronic phase display compromised lymphokine-activated killer (LAK) cell induction, which is partly restored after therapy with interferon . However, the relative resistance of the leukemic cells from these patients to autologous or allogeneic LAK lysis is not affected by this treatment. In an attempt to render CML cells more susceptible to lysis or cytostasis, they were precultured in serum-free medium with or without recombinant growth factors. In eight patients studied, interleukin-3 (IL-3) significantly enhanced the spontaneous short-term (6-day) proliferation of CML cells, with retention of ability to form colonies in methylcellulose. Culture in either medium alone or IL-3 led to a significant enrichment of CD14⁺ and CD33⁺ cells but to a reduction in CD34⁺ cells. In contrast, culture of the same cells in IL-2 (to generate autologous LAK activity) resulted in a loss of CD14⁺ and CD33⁺ as well as CD34⁺ cells but in a significant increase in CD3⁺ and CD56⁺ cells. Despite similarities in their phenotypes, IL-3 cultured cells but not those cultured in medium alone acquired susceptibility to lysis by the IL-2-cultured autologous LAK cells. These results may have significance for the design of novel combination immunotherapy in CML.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)     

Romanowsky dyes and Romanowsky-Giemsa effect

Summary A reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18 100 cm^–1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations.Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18 100 cm^–1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions.The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly orientated dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence. Subsequently, the absorption spectra of the highly orientated dye complexes were measured with plane polarized light. We found that the transition moments, m _AB, of the bound AB cations in DNA-AB and DNA-AB-EY are orientated almost perpendicular to k, i.e. m _ABk. On the contrary, the transition moments, m _EY, of the bound EY anions in DNA-AB-EY are polarized parallel to k, i.e. m _EY k. The transition moments m _AB and m _EY lay in the direction of the long axes of the AB and EY chromophores. For that reason, in both DNA-AB and DNA-AB-EY the long molecular axes of the AB cations are orientated approximately perpendicular to the DNA chains, while the long molecular axes of the EY chromophores are polarized in the direction of the DNA chains. Therefore, in DNA-AB-EY the long axes of AB and EY are perpendicular to each other, m _ABm _EY. This molecular arrangement fully agrees with our quantitative measurements and with the theory of the absorption of plane polarized light by orientated dye complexes, which has been developed and discussed in detail.     

Nerve growth factor: Cellular localization and regulation of synthesis

1. The role of nerve growth factor (NGF) as a retrograde messenger between peripheral target tissues and innervating sympathetic and neural crest-derived sensory neurons is supported by the observations that (a) the interruption of retrograde axonal transport has the same effects as the neutralization of endogenous NGF by anti-NGF antibodies and (b) the close correlation between the density of innervation by fibers of NGF-responsive neurons and the levels of NGF and mRNANGF in their target organs. 2. In situ hybridization experiments have demonstrated that a great variety of cells in the projection field or NGF-responsive neurons is synthesizing NGF, among them epithelial cells, smooth muscle cells, fibroblasts, and Schwann cells. 3. The temporal correlation between the growth of trigeminal sensory fibers into the whisker pad of the mouse and the commencement of NGF synthesis initially suggested a causal relationship between these two events. However, in chick embryos rendered aneural by prior removal of the neural tube or the neural crest, it was shown that the onset of NGF synthesis in the periphery is independent of neurons, and is controlled by an endogenous "clock" whose regulatory mechanism remains to be established. 4. A comparison between NGF synthesis in the nonneuronal cells of the newborn rat sciatic nerve and that in the adult sciatic nerve after lesion provided evidence for the important regulatory role played by a secretory product of activated macrophages. The identity of this product is currently under investigation.     

Artificial dimers of native actin: Preparation and properties in biological functions

With the aid of tartryl-bis--aminocaprylazide artificial dimers were produced from F actin from rabbit striated muscle. These derivatives will not polymerize by themselves but are able to copolymerize fully with native G actin. By modification of a single side chain per dimer, this copolymerization was completely inhibited. The dimers are able to activate subfragment I ATPase of myosin and bind to DNase I with inactivation of the enzyme in the same manner as native G actin. Within the dimer, one ADP is immobilized and will exchange against ATP extremely slowly. The dimers do not bind to the mushroom toxin phalloidin.     

Pleotropic mutants from Alcaligenes eutrophus defective in the metabolism of hydrogen,nitrate, urea,and fumarate

Chromosomal mutants of Alcaligenes eutrophus unable to grow with molecular hydrogen as the energy source also failed to grow with nitrate as the terminal electron acceptor or as a nitrogen source. The mutants (Hno^–) (i) formed neither soluble nor particulate hydrogenase antigens, (ii) expressed only about 50% the wild type level of ribulosebisphosphate carboxylase activity, and (iii) transported nickel, an essential constituent of active hydrogenase, at a significantly lower rate than wild type cells. Moreover, the mutants grew very slowly with urea as nitrogen source and did not express urease. Growth on formamide was also affected and formamidase activity was induced to only a very low level. Growth of the Hno^– mutants on succinate, glutamate, fumarate, and malate was significantly slower than wild type, and a reduced rate of succinate incorporation into the mutant cells was demonstrated. The highly pleiotropic phenotype of Hno^– mutants is indicative of a chromosomal gene with a considerable physiological importance. It affected the expression of both chromosomal and megaplasmid encoded systems of energy, carbon, and nitrogen metabolism. Thus, the hno mutation restricts the metabolic versatility but does not affect the basic metabolic functions of the organism.