Method of clonal micropropagation of different willow species and hybrids

Conditions of cultivation and micropropagation of selected biotypes of five willow species (Salyx dasyclados Wimm., S. caspica Pall., S. triandra L., S. purpurea L., and S. viminalis L.) and two hybrids (×S. acuminata S. and ×S. palustris Host.) were optimized. Data on in vitro propagation of S. caspica, S. triandra, S. purpurea together with hybrids S. acuminata and S. palustris were obtained for the first time. It has been demonstrated that the outcome of cultivation and propagation of willows strongly depends on genotypic peculiarities of initial plants. The optimal terms of isolation and sterilization of single-node segments for obtaining 50–75% of aseptic viable developing cultures were estimated. The nutritive media were selected providing induction of stem development (to 67%), their rooting (to 91%), elongation (to 3–6 cm), and multiplication (propagation coefficient of 4). The designed method (adopted to different genotypes) can be applied for obtaining aseptic in vitro cultures serving as initial plant material for genetic transformation and mass propagation of plants with new agriculturally valuable characteristics which are of interest for construction of bioenergetic plantations and for needs of the paper industry.     

Antibody-targeted photolysis: in vitro immunological, photophysical, and cytotoxic properties of monoclonal antibody-dextran-Sn(IV) chlorin e6 immunoconjugates.

A set of anti-melanoma immunoconjugates were prepared which contained chlorin e6: antibody molar ratios of 18.9:1, 11.2:1, 6.8:1, and 1.7:1. All immunoconjugates retained antigen binding activity regardless of the chromophore:antibody substitution ratio that was attained. In contrast, the ground-state absorption spectra of the immunoconjugates showed features which appeared to be dependent on the chromophore:antibody molar ratio. In addition, the quantum yield of singlet oxygen generated by the conjugated chromophores was observed to be significantly less than that observed with the unbound dye. Time-resolved absorbance spectroscopy of the chromophore excited triplet state indicated that the loss of singlet oxygen quantum yield resulted from diminished chromophore triplet yield. Analysis of data obtained from in vitro photolysis of target melanoma cells, in combination with that obtained from the immunochemical and photochemical studies, indicates that the observed immunoconjugate phototoxicity can be reasonably quantitatively represented by (1) the ability of the immunoconjugate to bind SK-MEL-2 cell surface antigen, (2) the amount of chromophore localized at the target cells by immunoconjugate binding, (3) the delivered dose of light at 634 nm, and (4) the singlet oxygen quantum yield of the antibody-bound photosensitizer. Though these data argue strongly for photolysis by the cumulative dosage of singlet oxygen at the cell membrane, nonetheless, the concurrent photoinduced release of other cytotoxic agents should not be ruled out.     

Specific silver staining of experimentally undercondensed chromosome regions

Treatment of human and mouse cell cultures with DNA binding AT-specific compounds and with some base analogues induced distinct undercondensations in several heterochromatic chromosome regions. All those heterochromatic regions undercondensed by AT-specific DNA ligands (distamycin A, DAPI, Hoechst 33258) could be heavily labeled with the silver(Ag)-staining technique; but the heterochromatic regions undercondensed with the cytidine analogue 5-azacytidine were Ag-negative. In metaphase chromosomes from BrdU-treated human cell cultures, the bifilarly substituted chromatids, which show a slight undercondensation, were also Ag-negative. Cytochemical analyses of the Ag-stained undercondensed heterochromatic regions showed that the Ag-stainable material consisted of nonhistone proteins. The mechanism of Ag staining in the undercondensed heterochromatic regions was compared with Ag staining of the nucleolus organizer regions.     

Electrophoretic analysis of the estrogen receptor. Molybdate stabilization and identification of the classical estrogen receptor

Conditions are defined which permit analysis of estrogen receptors from the mammalian uterus by polyacrylamide gel electrophoresis, thereby solving a longstanding problem encountered in previous attempts at such analysis, namely the failure of a large portion of the receptor population to enter such gels. A paramount requirement for entry of the estrogen-receptor complex into polyacrylamide gels is its maintenance in an untransformed state which does not form aggregates that are excluded from these gels. Of the multiple estrogen-binding proteins separated, only one (relative mobility of 0.5-0.6) possessed the definitive characteristics of the classical estrogen receptor. The inclusion of molybdate in extraction buffers selectively enhanced receptor recovery and facilitated its separation. Moreover, the estrogen-receptor complex so resolved is separated from other types of estrogen-binding proteins present in the uterine cytosol. These findings show that the molybdate-stabilized estrogen receptor exists in a single discrete form, but otherwise exhibits multiple forms that are probably artifactual. Electrophoresis in discontinuous buffers, but not in a continuous buffer system, promoted aggregate formation. This finding has implications concerning the subunit structure of the untransformed receptor.     

Comparative effects of various classes of mouse interferons on macrophage activation for tumor cell killing

The effects of mouse interferon-alpha (MuIFN-alpha), -beta (MuIFN-beta), and -gamma (MuIFN-gamma) on macrophage activation for tumor cell killing were determined by using proteose peptone-elicited peritoneal macrophages from C3H/HeN and C3H/HeJ mice under conditions that either included or were free of detectable endotoxin. Alone, under the conditions used, none of the interferons was able to activate macrophages directly for tumor cell killing. However, with a second signal provided to responsive macrophages by contaminating endotoxin, added bacterial lipopolysaccharide (LPS), or heat-killed Listeria monocytogenes (HKLM), all three types of interferon induced cytolytic activity, with MuIFN-gamma approximately 500 to 1000-fold more active than either MuIFN-alpha or -beta. Thus, all three interferons were able to prime macrophages for killing but required a second signal before cytolytic activity could be expressed. When MuIFN-gamma was mixed with either MuIFN-alpha or -beta and placed on macrophages, little or no killing developed. Mixtures of MuIFN-gamma with either MuIFN-alpha or -beta did increase the sensitivity of macrophages to triggering by LPS, however, compared with macrophages treated with MuIFN-gamma alone. The results are collectively important because they i) confirm that significant quantitative differences exist between the various interferons with regard to their capacity to prime macrophages for tumor cell killing; ii) indicate that to be an efficient activator each type of interferon must be combined with a second stimulus, such as LPS or HKLM; iii) show that neither MuIFN-alpha nor -beta can provide an efficient second triggering signal for macrophages that are primed by MuIFN-gamma; and iv) document that mixtures of MuIFN-gamma with either MuIFN-alpha or -beta are most efficient at inducing priming, compared with any one of the interferons used alone.