Rat embryos at the head-fold stage (9.5 days of gestation) were cultured for 32 hours in rat serum.Embryos rotated their axes (changing from the shape of a concave mid-region to that of a convex mid-region)during the last 5 hours of culture (from 27 h to 32 h in culture). Addition of 0.1 μg/ml cytochalasin D to the culture medium for this 5-hour period prevented axial rotation in the embryos and disturbed the appearance of microfilaments in the dermatome, the dorsal region of the trunk neural tube, and the dorsal epidermis.During the period of axial rotation, the dermatome and the dorsal epidermis extended and showed the arrangement of microfilaments along the cranio-caudal axis in the control embryos but not in the treated embryos. The dorsal region of the trunk neural tube in the control embryos consisted of a seam of neuroepithelial cells in which microfilaments were apparently arranged along the cranio-caudal axis but the region in the treated embryos was crowded with the neuroepithelial cells piled up randomly and microfilaments showed no arrangement.These results suggest that changes in the shapes and arrangement of the cells in the dermatome, the dorsal region of the trunk neural tube, and the dorsal epidermis cause extension of these tissues along the cranio-caudal axis and result in axial rotation. Microfilaments may play an essential role in changes in the shapes and arrangement of the cells in these tissues.

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Human promyelocytic leukemia cell line, HL-60, is known to proliferate exponentially in a serum-free synthetic medium supplemented with insulin and transferrin (BREITMAN, T.R. et al. (1980). Exp.Cell Res., 126 : 494-498). Among four HL-60 cell lines tested in this medium (serum-free medium), however, a cell line, HL-60/Biken ceased to proliferate after two days culture and most of the cells died within a week. Addition of purified serum lipoprotein (LDL or HDL)to the serum-free medium almost completely restored the proliferating activity of the cells. Total lipids extracted from the lipoproteins could replace the lipoproteins in promoting cell proliferation. Among various lipid components of the lipoproteins, only cholesterol showed a high stimulatory effect on cell proliferation, whereas other lipids tested were ineffective, except for sphingomyelin, cerebroside, and phosphatidic acid which showed limited stimulatory effects. As for the intermediates of cholester l biosynthesis, desmosterol was also effective, whereas lanosterol was rather inhibitory. Chromatographic analyses of the lipids synthesized by HL-60/Biken cells and wild type cells (HL-60/RCB) cultured in serum-free medium, clearly demonstrated that, in HL-60/Biken cells, cholesterol synthesis was almost completely blocked and lanosterol was accumulated 10-fold that in wild type HL-60/RCB cells. All of these results indicate that HL-60/Biken is a variant cell line defective in cholesterol synthesis in the process synthesizing desmosterol from lanosterol. The variant HL-60 cells showed a marked resistance to cell differentiation along both monocytic and granulocytic pathways when compared with wild type HL-60 cells. The cell line may be useful for the study of the role of cholesterol in cell differentiation.

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A primary culture of undigested fat tissue fragments was used to obtain fat cells in vitro. On day 2 of culture, immature fat cells, which are fibroblast-like fat cells containing fine lipid droplets, appeared, surrounding the fat tissue fragments, and began to proliferate extensively. Afterwards, these fibroblast-like fat cells grew to become multilocular fat cells containing larger intracytoplasmic lipid droplets, and differentiated furtherinto unilocular fat cells containing a single large intracytoplasmic lipid droplet.
Treatment with dibutyryl-cAMP, which is a second messenger of the lipolytic factor, caused the cultured fat cells to retract, and the intracytoplasmic lipid droplets of those fat cells became finely granulated and decreasedalong with an increase of hormone-sensitive lipase activities. Conversely, administration of insulin caused the lipid droplets in the fat cells to increase and become larger along with an increase of α-glycerophosphate dehydrogenase activities. These findings indicate the occurrence of lipolysis and lipogenesis of fat cells in vitro.
Immuno-cytochemistry revealed that vimentin surrounded intracytoplasmic lipid droplets, and became distinct with an increase of lipid droplets through lipogenesis in the fat cells. Vimentin seems to be correlated to the behavior of lipid droplets in the fat cells.
Fat cells in this study showed the appropriate cellular structures and functions in response to stimulation of lipolysis and lipogenesis under culture conditions. It is expected that in vitro culture of fat cells will facilitate cell biological elucidation of obesity in the future.

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To determine the functional significance of endoplasmic reticulum chaperones in hematopoietic cells, we analyzed the expression and post-translational modification of BiP/GRP78 and GRP94 as well as the cytoplasmic chaperones HSP70 and HSC70 during the differentiation of a mouse myeloid leukemia cell line, M1. The amounts of BiP/GRP78 and GRP94increased several-fold when M1 cells were induced to differentiate into macrophage-like cells by treatment with interleukin-6 (IL-6). Synthesis began to increase at 4 hr after IL-6 treatment. The phosphorylated form of BiP/GRP78 increased during the later stages of differentiation. These data suggested that the chaperone activity of BiP/GRP78 and GRP94 may be needed for differentiated macrophage-like cells or for the differentiation event itself, and that functionally different BiP/GRP78 accumulate during the differentiation of M1 cells.

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Human fibroblasts (KMST-6/RAScell line), which was malignantly transformed in vitro with ⁶⁰Co gamma-rays and the c-Ha-ras oncogene, produced a large amount of granulocyte colony-stimulating factor (G-CSF). The production was greater during the logarithmic growth phase than during the stationary phase.cAMP and theophylline, alone or in combination, and butyrate significantly enhanced G-CSF production, but dexamethasone or 5-azacytidine did not. Enhanced production of G-CSF by these agents was regulated at the posttranscriptional level. Neither the expression of the ras oncogene nor the tumorigenicity of the cells correlated with the production of G-CSF.

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We used immunocytochemical methods and a specific antibody to identify proliferating cell nuclear antigen (PCNA)in Drosophila embryos during the postblastoderm cell division cycles. The strong nuclear staining observed during interphase disappeared at prophase, staining remained nil throughout the remaining mitotic (M) phases and reappeared in nuclei at the next interphase. As the embryos with the homozygous string^-mutation sustained the strong staining signal in nuclei throughout embryonic development, disappearance of the staining signal probably depends on string function and is coupled with the onset of mitosis. When cells in embryos were arrested at M phase following treatment with TN-16 or Vinblastine, the staining signal with the anti-PCNA antibody was lost. However, Western blots showed that the level of PCNA protein in M phase-arrested embryos did not decrease. Therefore, the disappearance of staining signals is apparently due to reorganization of PCNA protein in the multiprotein complex in nuclei, rendering it inaccessible to the antibody, rather than to the degradation of PCNA protein. In contrast to findings in developing embryos, cultured Drosophila Kc cells stained strongly with the anti-PCNA antibody, during both interphase and the M phase. In genetic crossing experiments of transgenic flies carrying the lacZ gene under the control of the PCNA gene regulatory region (-607 to + 137 with respect to the transcription initiation site) with string^- mutant flies, the PCNA gene promoter seems to function in a manner independent of cell cycle progression or of functions of the string gene.

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We described the initial events in the interaction between an oligodendrocyte process and an axon in culture utilizing video time-lapse microscopy. Myelination of an axon by the lamellipodium of an oligodendrocytewas achieved in several steps of cellular process development and coordinated interaction between axon and oligodendrocyte. The initial stage of contact included the formation of a lamellipodium process at the end of an oligodendrocyte process. It appeared that this process contacted the axon several times and was then retracted, and that the filopodia and lamellipodium underwent morphological changes prior to the onset of the myelination.In the second stage, the lamellipodium appeared to thicken and anchor to the axon. Finally, when rippling of the lamellipodial ruffling occurred, the angle between the anchoring filopodium and the axon changed depending on the direction of lamellipodial movement, and the lamellipodium, which was folded in layers, wrapped around the axon like a transverse wave in one motion as observed on the video screen. Thereafter, the lamellipodiumassumed a "bursting" form within minutes in real time. This is the first comprehensive overview of howan oligodendrocyte plasma membrane wraps around an axon to form myelin.

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We studied the mechanism of the lidocaine-induced shape change in human erythrocytes. Immunohistochemical analysis of erythrocytes using spectrin-specific antibodies revealed aggregation of fluorescence in lidocaine-treated cells, while the fluorescence was distributed diffusely in untreated cells. The intracellular pH in lidocaine-treated erythrocytes was examined by flow cytometry of the cells labeled with 3'-aeetyl-2'-carboxyethyl-6', 7'-(dihydropyran-2'-one)-5-carboxyfluorescein diacethoxymethylester (BCECF-AM), and was found to decrease with increasing concentrations of lidocaine. Pre-treatment of erythrocytes with acetazolamide, an inhibitor of carbonic anhydrase, inhibited the lidocaine-induced spectrin aggregation and decrease in intracellular pH. When erythrocytes were incubated in medium containing bafilomycin A₁, an inhibitor of V-ATPase, followed by incubation with lidocaine, the cells changed shape slightly and the intracellular pH showed a small decrease in comparison with control. Spectrin dimers extracted from membranesof normal erythrocytes were incubated in buffers of various pHs and analyzed by SDS-PAGE. The amounts of spectrin dimers and tetramers decreased, while that of oligomers increased with decreasing pH. These results suggest that the lidocaine-induced shape change in human erythrocytes may occur by the conformational change of spectrin in a process that may be mediated by carbonic anhydrase and activation of V-ATPase.

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Contact reaction is the morphological expression of allogeneic recognition by hemocytes in the ascidian, Halocynthia roretzi; namely they undergo an exocytotic burst upon contact with non-self (allogeneic) hemocytes in vitro. We have found that contact reaction is accompanied by a burst of cyanide-insensitive oxygen consumption that, unlike exocytotic events of mammalian phagocytes, is Ca²⁺ -dependentand does not culminate in the production of superoxide anions or H₂O₂. Instead, the burst is due to the release of phenoloxidase from hemocytes as known for the self-defense systems in insects and crustaceans. The activity of phenoloxidase released from hemocytes corresponds well to the degree of contact reaction observed under the microscope.Therefore, it is possible to quantify the contact reaction simply by measuring the activity of phenoloxidase released from hemocytes into the medium.

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Induction of mitochondrial serine : pyruvate aminotransferase (SPT) in rat liver by administration of glucagon was studied quantitatively by immunoblot analysis and cryoimmunogold electron microscopy.Immunoblot analysis revealed that two daily injections of glucagon produced marked increase of SPT protein mass to a level as much as 18 times that of the untreated rat. Cryoimmunogold electron microscopic analysis showed that the labeling density of the mitochondria increased in a parallel manner. Thus the induction of SPT analyzed by two methods showed an excellent correlation with a relative correlation coefficient of 0.98, indicatingthat the induction of SPT can be analyzed quantitatively by immunogold electron microscopy on cryoultrathin sections.

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We report here the in vitro culture of the atrial epithelium, which is the major formative tissue of the budding tunicate, Polyandrocarpa misakiensis. Preliminary studies suggested that both pH and osmotic pressure of the basic seawater medium should be lowered significantly (pH 6.8, 800-830 mOSM). In the growth medium consisting of modified millipore-filtered seawater, Dulbecco's modified Eagle medium and 3% fetal bovine serum, cells spread out from the epithelial explant and proliferated with a doubling time of about 13 hours. They could be cloned and cultured successively. They contained the Polyandrocarpa lectin gene, showing that they were indeed of tunicate origin. At a low cell density (< 3x10² cells/mm²), clonal cells took a spherical form and contained several granules in the cytoplasm. At a high cell density (>3x 10⁴ cells/mm²), on the other hand, they gave rise to smaller cells without any specialized features and, finally, to dark flattened cells. Consistent with this observation, con fluent cells lost the atrial epithelium-specific antigen, which reappeared on the cell surface when they were re-plated at a low density. In conclusion, we have established for the first time tunicate cell lines. They appeared to differentiate and dedifferentiate repeatedly in our culture system.

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Human diploid fibroblasts (HDF) have a limited in vitro lifespan of population doubling level (PDL) 50-70. The molecular mechanism underlying cellular senescence and immortalization is not thoroughly understood. It has been reported that the cytoskeleton has diverse functions and may have a role in growth regulation through association with other cellular components.
To shed light on the relationship between functions of the cytoskeleton and senescence or immortalization, We investigated the alterations in gene expression after immortalization and measured the amounts of mRNAs for talin, vinculin, α-actinin, tropomyosin 1 (TM1), vimentin, lamin A and C, and α-tubulin by slot blot and Northern blot analyses. We found that the mRNAs for vinculin and vimentin were reduced and the mRNA for lamin A was increased in immortalized cells.
We also studied the cytoskeletal protein levels and their intracellular distributions by Western blot analysis and immunostaining. Most of the proteins studied behaved in a way similar to the mRNAs through senescence and immortalization. Vinculin, tropomyosins and vimentin showed their altered distributions in immortalized cells.

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