Functional alterations in beta''-actin from a KB cell mutant resistant to cytochalasin B

We recently described the isolation of mutant KB cells (Cyt 1 cells) resistant to the cytotoxic effect of cytochalasin B (CB). This mutant carried an altered beta-actin; i.e., beta'-actin (Toyama, S., and S. Toyama. 1984. Cell. 37:609-614). In the present study, we have examined the functional properties of actin in Cyt 1 cells. Our results showed that increased resistance of Cyt 1 cells to CB was reflected in altered properties of beta'-actin itself. This was shown directly by two findings. First, the polymerization of beta'-actin was more resistant than that of beta- or gamma-actin to the multiple effects of CB. Second, beta'-actin bound less CB than beta- or gamma-actin. The functional alteration of beta'-actin in Cyt 1 cells was further supported by the observation that, although treatment of KB cells with CB increased the pool of unpolymerized actin, the same treatment did not affect the pool of unpolymerized actin in Cyt 1 cells, and that microfilaments of Cyt 1 cells were more resistant to the disrupting action of CB than those of KB cells. These results strongly suggest that the primary site of action of CB on cell motility processes is actin.     

Characterization of membrane-bound spermidine dehydrogenase of Citrobacter freundii.

Spermidine dehydrogenase found in the membrane fraction of Citrobacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, 1,3-diaminopropane and gamma-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.     

Cure of osteopetrosis by injection of allogenic bone marrow into "op" rats treated with cyclosporin A

The cure of osteopetrosis by allogenic bone marrow injection has been obtained in "op" mutant rat kept under cyclosporin A treatment. This immunosuppressive agent able to prevent the rejection of transplanted cells does not impair the propriety of these cells to restore the bone resorption process in this severe osteopetrotic form.     

Effects of colcemid and taxol on microtubules and intermediate filaments in chick embryo fibroblasts

Reports on how changes in microtubule (MT) distribution or polymerization affect the distribution of intermediate filaments (IFs) differ. Therefore, we have used cytoimmunofluorescence techniques and electron microscopy to systematically examine and compare the arrangements of MTs and IFs in cultures of chick embryo fibroblasts under the following conditions: at different times during the cell cycle, in the presence of Colcemid or of taxol, in the presence of both drugs in succession or simultaneously in varying ratios, and during recovery from treatment with Colcemid or taxol. We have found that depolymerization of MTs by 1 microM Colcemid resulted in the rapid formation of massive IF-cables, structures distinct from "collapsed IFs" or "juxtanuclear coils." Neither the rapid formation of IF-cables nor their dispersion during recovery required protein synthesis. Cells treated with 10 microM taxol rapidly formed MT-bundles, as well as aggregates of intertwining IFs, termed "IF-skeins." MT-bundles and IF-skeins displayed strikingly complementary distributions. This reciprocal distribution of packed MTs and IFs was also obvious in untreated anaphase and telophase cells. When 10 microM taxol and 1 microM Colcemid were applied simultaneously, the complementary distributions of MT-bundles and IF-skeins mimicked those in taxol alone. This ability of taxol to block Colcemid's effects was concentration dependent. Decreasing the taxol: Colcemid ratio allowed the depolymerization of MTs, which correlated with the formation of IF-cables.     

Factors affecting formation of micronucleus-like structures after colchicine treatment of Trichoderma reesei

Micronucleus-like structures were produced in Trichoderma reesei only when 0.1% colchicine treatment was used to enhance nuclear division. The average DNA content of these small nuclei was 30% that of the normal nuclei, indicating that they were aneuploid nuclei. Such small nuclei may be useful in transferring small amounts of DNA into protoplasts.The authors are with the Department of Food Technology, Faculty of Horticulture, Minamikyushu University, Takanabe-Cho, Hibarigaoka, Miyazaki 884, Japan;     

Effects of 12-O-tetradecanoyl-phorbol- 13-acetate on myofibril integrity and Ca2+ content in developing myotubes

The cocarcinogen 12-O-tetradecanoyl-phorbol-13-acetate (TPA) has a prompt and selective effect on striated myofibrils in well-formed, multinucleated myotubes. The myofibrillar apparatus is totally disrupted and largely degraded after 3 days in TPA. Fluorescent-tagged antibodies to muscle-specific light meromyosin and electron microscopy document this catabolic effect of TPA. This selective degradation of fully assembled striated myofibrils is readily reversible. This reassembly of myofibrils requires de novo protein synthesis. The TPA-treated myotubes are unusually rich in autophagosomes, organelles rarely observed in normal myotubes. TPA has no discernable effect on the morphology of the subsarcolemmal microfilaments, mitochondria, microtubules, or sarcoplasmic reticulum (SR). The programmed disappearance of the fibroblast-type, intermediate 10-nm filaments (FIF) that occurs as normal myotubes mature is not altered by TPA. However, the TPA-treated myotubes depleted of myofibrils are filled with an extensive meshwork of the muscle-type intermediate 10-nm filaments (MIF). The drug has no obvious effect on the constitutive contractile proteins comprising the submembranous microfilaments, or the FIF in presumptive myoblasts or fibroblasts, nor does it affect the motility associated with these replicating cells. The striking increase in total calcium content which occurs as normal myotubes mature is absent in myotubes treated with TPA.     

Establishment of a variety of human bone marrow stromal cell lines by the recombinant SV40-adenovirus vector.

Various human bone marrow stromal cell lines were established from the adherent cell populations by introduction of the recombinant SV40-adenovirus vector with an infection or electric poration procedure. As compared with DNA transfection, the vector introduction was able to immortalize the cells with more than 100 times higher efficiency. Morphological and cytochemical analyses indicated that various cloned cell lines with different properties were isolated by the vector introduction. All the established cell lines expressed SV40 large T antigen. These results provided the evidence indicating that the recombinant SV40-adenovirus vector was a useful tool to establish a variety of cell lines with different biological activities from human bone marrow stroma.     

Measurement of voltage dependence of capacitance of planar bilayer lipid membrane with a patch clamp amplifier.

The voltage dependence of capacitance was measured by using the setup which was almost the same as that for the study of ion channels. The coefficient which represents the voltage dependence of capacitance itself also changes as a function of the duration of voltage application if hexadecane is contained in bilayer lipid membrane (BLM). The method of Alvarez, O., and R. Latorre (1978. Biophys. J. 21:1-17) was extended to treat BLM with hexadecane.