We examined the effects of large T antigen of simian virus 40 (SV40) on the proliferation phenotypes of temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, which cease proliferating in the G1 phase of the cell cycle at a restrictive temperature (39.8°C). Four ts mutants, each representing independent complementation groups, were transformed with the dl-884 mutant of SV40 which lacks the unique coding region for small t antigen. In the case of two ts mutants, their transformed derivatives did not cease proliferation at 39.8°C. In the other two mutants, the transformed cells continued to enter the S phase but the cells became detached from the dishes thereafter, at 39.8°C.The proliferation phenotypes of the dl-884-transformed cells at 39.8°C were quite similar with those of the same mutants transformed with the wild-type SV40. These results indicate that large T antigen alone is sufficient to overcome the inhibition of cellular entry into S phase caused by four different ts defects and determines the proliferation phenotypes of the cells after entering the S phase at a restrictive temperature, and that small t antigen does not alter the cellular phenotypes determined by large T antigen.

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Transmission electron microscopy was used to analyse the process of interaction of Trypanosoma cruzi with resident and activated mouse peritoneal macrophages. Initially, the parasites are located within a membrane-bounded endocytic vacuole. Lysosomes from the host cell fuse and discharge their content into the parasite-containing vacuole, as visualized by localization of horseradish peroxidase and acid phosphatase activity. Acridine orange was used to label secondary lysosomes in order to quantify the process of lysosome-phagosome fusion by fluorescence microscopy. The fusion index was higher for amastigote than for epimastigote and trypomastigote forms. Images were obtained showing that a few hours after ingestion of trypomastigote forms by the macrophages there is progressive disruption of the membrane lining the vacuole, until its complete disappearance.

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The growth of capillaries from mouse bone marrow was studied in collagen gel. When the culture contained sufficient bone marrow cells (more than 1 × 10⁶ cells) and cell clusters, short capillaries with lumina appeared about one to two weeks after inoculation, following the proliferation of fibroblastic cells and hemopoietic cells. Four weeks after inoculation, these capillaries formed a network among hemopoietic cells and adipocytes.
Electron microscopic observations revealed that these capillaries had thin walls and poorly developed basement membranes, similar to the sinusoids of bone marrow. These capillaries did not appear when the amount of inoculated bone marrow was reduced or dispersed to the point that the marrow cell clusters disappeared. The quantity of the inoculum and the clustering of cells, therefore, seems to play important roles in the appearance of the capillaries.

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The effects of hydroxyurea (HU) and thymidine (TdR) on cell cycle progression in V79 Chinese hamster lung cells were examined by flow cytometry. Suppression of the cell cycle progress rate by HU was further enhanced by the combination of a low concentration of TdR and HU as com-pared to that induced by TdR alone; i.e., these drugs were shown to have a synergistic effect. It was concluded that the presence of TdR was effective in assisting HU-induced suppression of DNA synthesis.

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The localizatioin of alpha-actinin, tropomyosin, myosin and actin in odontoblasts was examined by fluorescence microscopy using well characterized antibodies and rhodamine-phalloidin. All the reagents labeled the distal end of the cell body in the form of an oval ring with a preferential axis along the tooth axis. This ring was often interrupted. In conventional electron microscopy, microfilament bundles with periodical dense spots were running along the tooth axis at the level of the distal end of the cell body. The periodicity was about 0.6-1.0 μm. It may be possible that this dynamic structure functions to keep odontoblasts in a layer by contracting in an isometric form.

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A filamentous structure in the apex of the acrosome, termed "truncated cone", was isolated from abalone sperm heads, and its structural component was characterized. Observation of the isolated truncated cone fraction by negative staining revealed that the truncated cone was composed of a helically coiled filamentous structure which was resistant to treatment with non-ionic detergent and high salt solutions. SDS PAGE of the isolated truncated cone fraction revealed 3 major polypeptides of 60, 86, and 200 kD. Im-munofluorescence microscopy and immunoelectron microscopy using affinity-purified monospecific antibody against the 60 kD protein revealed that the 60 kD protein was localized exclusively to the truncated cone in both the isolated truncated cone fraction and the acrosome-reacted sperm. Immunoblotting analysis showed that anti-vimentin antibody cross-reacted with this 60 kD protein. These results suggest that the 60 kD protein is a component of the truncated cone, which shares antigenic determinants with vimentin.

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Breakdown of cortical granules was investigated by microinjecting Ca-EGTA buffers at various concentrations of Ca²⁺, inositol 1, 4, 5- trisphosphate (IP₃), guanosine triphosphate (GTP) and guanosine 5' -O- thiotriphosphate (GTPγS) into echinoderm (Clypeaster japonicus, Temnopleurus hardwicki, and Scaphechinus mirabilis) eggs using a video microscope. The breakdown started in the region near the injection site and then propagated over the entire surface of the egg. The cortical granules did not synchronously break down side by side, but instead underwent random breakdown, in the same manner as that upon fertilization. The reaction times of the breakdown of the cortical granule after the injection of Ca-EGTA buffers, IP₃, and GTPγS were 0.4 sec, 1.0 sec, and 2.7 sec, respectively, in C. japonicus eggs, and were in the same order in T. hardwicki and S. mirabilis eggs. It is inferred from this order of reaction times that GTP-binding proteins, IP₃ and Ca²⁺ act in this sequence upon fertilization in echinoderm eggs.

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Human recombinant interferon, (rIFN)-γ, induced a human neuroblastoma cell line GOTO to differentiatte, but neither rIFN-αA nor -β did. To elucidate the mechanism of this rIFN-γ-specific differentiation-inducing effect, we established two rIFN-γ-resistant variant GOTO clones. They were insensitive to the growth-inhibitory and differentiation-inducing effect of 1 × 10³ IU/ml rIFN-γ. They were slightly sensitive to the growth-inhibitory effect of rIFN-γ (2 × 10⁴ IU/m1). Parental GOTO cells were very insensitive to the antivesicular stomatitis virus (VSV) effect of all three types of rIFNs and even 2 × 10⁴ IU/ml rIFN-γ could not inhibit the cytopathic effect of 5 TCID₅₀ VSV by 50%. The degree of this insensitivity was the same in the variant GOTO cells as in the parental GOTO cells.

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Involvement of the Ca²⁺ ion and membrane potential in the regulation of the Na⁺-dependent glycine transport was investigated in the Chang liver cell. Depolarization of the cell membrane was associated with the uptake of glycine, showing a half-maximal change in the potential at 1.7 mM of glycine, a value corresponding to the Km for the glycine transport. Addition of EGTA to the assay medium inhibited the glycine uptake in a dose-dependent manner in accordance with the decrease of the intracellular free Ca²⁺ level and the extent of depolarization of the cell membrane. Trifluoperazine inhibited the glycine uptake with a half-maximal effect at about 16μM, a concentration giv-ing a half-maximum of membrane depolarization. Quinine, a blocker of the Ca²⁺-dependent K⁺ channel in liver cells, inhibited the glycine transport and depolarized the cell membrane, depressing K+ release from the cells. The glycine uptake was stimulated by the addition of a calcium ionophore (A23187) to the assay medium, but the uptake was extremely depressed by addition of quinine to the medium or by pretreatment of cells with trifluoperazine, to an extent as low as half of the control. Concomitantly, the K⁺ release increased by A23187 was depressed in the corresponding ratio by addition of quinine to the medium. These results support the concept that the Na⁺-dependent glycine transport is regulated by the cytosolic free Ca²⁺ concentration through the change of the cell membrane potential which is modulated by the activity of the Ca²⁺-dependent K⁺ channel requiring calmodulin as a regulator.

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The stimulatory effects of lymphokines, interleukin 3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4), and the inhibitory effects of transforming growth factor β (TGF-/β) and the pertussis toxin, islet activating protein (LAP), on multi-factor-dependent myeloid cell lines were examined. The effects of IL-3 on a mast cell progenitor clone, IC2 were indistinguishable from those of GM-CSF with respect to their concentration-response curves for induction of DNA synthesis and capability to maintain cell growth for many months. IL-4 acts differently on IC2 cells: the maximum level of DNA synthesis induced by IL-4 is always lower than that in-duced by IL-3 or GM-CSF and IL-4-induced proliferation is transient. IL-4, however, synergistically induced DNA synthesis of IC2 cells with limiting con-centrations of IL-3 or GM-CSF. When IC2 cells were cultured with saturating concentrations of IL-3, GM-CSF or a combination of both, the doubling time was 25±1 h, whereas it decreased to 17±1 h when IL-4 was further added to the cultures. IAP reduced the DNA synthesis of IC2 cells induced by the above three growth factors. The doubling time of IC2 cells was 30±2 h when IC2 cells were cultured with sufficient concentrations of IL-3 in the presence of IAP. Cell cycle analysis revealed that the fraction of cells in Gl was decreased by IL-4 but was increased by IAP. TGF-/3 also reduced IL-3-dependent DNA synthesis and increased the fraction of cells in Gl. The inhibitory effect on IL-3-dependent growth of IC2 cells was not increased when these cells were exposed simultaneously to TGF-β and IAP. These results suggest that IL-3 and GM-CSF stimulate the growth of IC2 cells through similar pathways and that IL-4 augments the action of IL-3 or GM-CSF by decreasing the Gl period. It is also suggested that IAP and TGF-β retard the growth of IC2 cells by increasing the fraction of cells in Gl.

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The effects of cell contacts and the attachment of cells to the substratum on growth-factor-induced survival of 3T3-L1 cells were investigated to clarify their involvement in the maintenance of cell viability. When 3T3-L1 cells in low-density cultures or in high-density cultures were harvested with EDTA solution and cultured in the absence of calf serum, almost all cells from the low-density cultures lost viability 24 h later. However, about 15% of the cells harvested from high-density cultures survived for 24 h in the absence of calf serum. Addition of calf serum also enhanced the survival of cells from high-density cultures to a much greater extent than that of cells from low-density cultures. Addition of fibroblast growth factor enhanced the survival of cells, especially in the case of cells from high-density cultures. However, epidermal growth factor and platelet-dervied growth factor failed to enhance survival. Coating of culture dishes with vitronectin slightly enhanced cell survival. Addi-tion of fibroblast growth factor markedly enhanced the survival of cells on the dishes coated with vitronectin or with fibronectin, but not on the dishes coated with heat-denatured bovine serum albumin. These results suggest that fibroblast growth factor promotes survival of 3T3-L1 cells, depending on cell-to-cell contacts during prior culture and on the adhesion of cells to the substratum.

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Phagocytosis of fluorescent microspheres in V79 cells arrested in the S phase by hydroxyurea (HU) was investigated by flow cytometry. The phagocytic activity increased with the exposure time of HU. No dependency on the HU concentration was observed in the concentration used. Cell size (projec-tion area) and membrane fluidity, two factors effected by HU treatment, were examined in relation to phagocytic activity, and were found to be significantly increased. The elevation in phagocytic activity could be interpreted in terms of the alteration in the two physiological factors.

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Recombinant λ. phage DNA, encapsulated in phage particles and coprecipitated with calcium phosphate, efficiently transforms cultured mammalian cellswithout a requirement for carrier DNA. The present paper analyzes the stability of the transformants obtained by the phage transfer method. λ. phage particlescontaining recombinant DNA that includes the thymidine kinase (TK) gene of herpes simplex virus type 1 as a selective marker were introduced into Ltk- cells deficient in TK activity, and TK⁺ transformants were selected in HAT medium. To test the stability of the TK⁺ phenotype of the transformants, seven individual transformant clones were isolated, cultured in HAT selective medium and then in non-selective medium for various lengths of time. After such culture, transformants were allowed to develop colonies in both selective and non-selective medium. For all seven transformant clones, the numbers of colonies obtained in the two types of medium were almost identical, irrespective of whether or not each transformant clone had been previously cultured for 15 to 50 days in non-selective medium. This result suggests that most transformants obtainedby the phage transfer method maintain the TK⁺ phenotype stably, for at least 50 days, when grown in non-selective medium.

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