Heterochromatin protein (HP)1γ is not only in the nucleus but also in the cytoplasm interacting with actin in both cell compartments

Confocal and electron microscopy images, and WB analysis of cellular fractions revealed that HP1γ is in the nucleus but also in the cytoplasm of C2C12 myoblasts, myotubes, skeletal and cardiac muscles, N2a, HeLa and HEK293T cells. Signal specificity was tested with different antibodies and by HP1γ knockdown. Leptomycin B treatment of myoblasts increased nuclear HP1γ, suggesting that its nuclear export is Crm-1-dependent. HP1γ exhibited a filamentous pattern of staining partially co-localizing with actin in the cytoplasm of myotubes and myofibrils. Immunoelectron microscopic analysis showed high-density immunogold particles that correspond to HP1γ localized to the Z-disk and A-band of the sarcomere of skeletal muscle. HP1γ partially co-localized with actin in C2C12 myotubes and murine myofibrils. Importantly, actin co-immunoprecipitated with HP1γ in the nuclear and cytosolic fractions of myoblasts. Actin co-immunoprecipitated with HP1γ in myoblasts incubated in the absence or presence of the actin depolymerizing agent cytochalasin D, suggesting that HP1γ may interact with G-and F-actin. In the cytoplasm, HP1γ was associated to the perinuclear actin cap that controls nuclear shape and position. In the nucleus, re-ChIP assays showed that HP1γ-actin associates to the promoter and transcribed regions of the house keeping gene GAPDH, suggesting that HP1γ may function as a scaffold protein for the recruitment of actin to control gene expression. When HP1γ was knocked-down, myoblasts were unable to differentiate or originated thin myotubes. In summary, HP1γ is present in the nucleus and the cytoplasm interacting with actin, a protein complex that may exert different functions depending on its subcellular localization.     

Absence of a correlation between cyclic nucleotide fluctuations and cell cycle progression

The levels of cAMP and cGMP were determined throughout the mitotic cycle of two independent strains of the naturally synchronous slime mold, Physarum polycephalum. The normal range of values was approx. 0.5–2.5 pmoles cAMP/ mg protein and 0.01–0.08 pmoles cGMP/mg protein. In our standard laboratory strain, there was no systematic pattern to the variations in the values within the normal range and no unique time in the cycle when values significantly above normal were noted. In the other strain recently cultured from spherules, elevated levels of cGMP, but not cAMP, were observed in late G2 and in the S phase. The data suggest that elevated levels of these cyclic nucleotides are not required for normal progression of the Physarum mitotic cycle which is unperturbed by artificial synchronizing procedures.     

Polyol accumulation by two filamentous fungi grown at different concentrations of NaCl

The accumulation of polyols by Aspergillus niger (van Tiegh) strain S 1 and Penicillium chrysogenum (Thom) strain S 30 was followed during growth in media of different concentrations of NaCl. The major polyols found were glycerol, erythritol and mannitol. The total polyol pool increased in both organisms in response to raised salinity, and the proportion of glycerol and erythritol was markedly enhanced at high salinity.     

Accelerated HER-2 degradation enhanced ovarian tumor recognition by CTL. Implications for tumor immunogenicity

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369–377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.     

AMG531 stimulates megakaryopoiesis in vitro by binding to Mpl

Thrombopoietin (TPO) plays a pivotal role in megakaryopoiesis. TPO initiates its biological effects by binding to its receptor Mpl. A recombinant protein consisting of a carrier Fc domain linked to multiple Mpl-binding domains was constructed, and is called AMG531. To define the biological activity of AMG531, we examined the ability of AMG531 to support CFU-Meg growth and to promote megakaryocyte maturation in vitro. AMG531 stimulates CFU-Meg growth in a dose-dependent manner, and acts in concert with erythropoietin, stem cell factor, interleukin-3, and interleukin-6 to enhance CFU-Meg growth, similar to parallel experiments with TPO. AMG531-stimulated serum-free liquid cultures support the development of mature polyploid megakaryocytes with a predominant DNA content of 32 N and 64 N, identical to that of parallel TPO-stimulated cultures. Competitive binding experiments show that AMG531 effectively competes with 125I-TPO for binding to BaF3-Mpl cells or normal platelets. Treatment of BaF3-Mpl cells with AMG531 or with TPO resulted in rapid tyrosine phosphorylation of Mpl, JAK2, and STAT5. These results indicate that AMG531 is a potent stimulant of megakarypoiesis in vitro, and provide support for its further characterization in vivo.     

Characterization of bimodal cell death of insect cells in a rotating‐wall vessel and shaker flask

In previous publications, we reported the benefits of a high‐aspect rotating‐wall vessel (HARV) over conventional bioreactors for insect‐cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker‐flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first‐order term. Cell‐cycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 ± 1% to 24 ± 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell–cell interactions, waste accumulation, and mass transport. These studies may benefit insect‐cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 14–26, 1999.