A cell line (HuL-1) derived from normal fetal human liver was adapted to grow continuously in a modified Eagle's minimum essential medium without serum or hormones. The population doubling time of this adapted cell line (HuL-1-317) was about 72 h and the modal number of chromosomes was 54. The morphology of HuL-1-317 cells was round in the absence of serum, but at 37°C with the addition of serum (1-10%), the cells flattened. HuL-1-317 cells had a low level of alkaline phosphatase activity. However the enzyme activity was slightly enhanced by the combination of prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and a hypertonic concentration of NaC1 after 3 days of incubation at 37°C. The increase in alkaline phosphatase activity with the four agents was further amplified dose-dependently by the pretreatment of the cells with serum. The stimulatory effect of the serum was evident at concentrations as low as 1%, and was maximal at 20%. The half life of the effect of serum on alkaline phosphatase induction was 48 h at 37°C. Serum alone could not enhance the enzyme activity without the four agents. The present results indicate that serum contributes to the regulation of alkaline phosphatase induction by the combination of prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCl in fetal human liver cells (HuL-1-317).

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This paper describes a method which enables the simultaneous measurement of both the concentration of cell surface receptors and the DNA content of individual lymphoid cells. Cells fixed with PLP (periodate-lysine-paraformaldehyde) were treated with ribonuclease (RNase). Transferrin receptors were then successively bound with monoclonal antibody against them and FITC-labeled antibody against the monoclonal antibody. Cells thus treated were stained with propidium iodide and two-parameter flow cytometric analysis was carried out.
Using this method, the expression of transferrin receptors on lymphoid cells was analyzed in relation to the action of T-cell growth factor (IL 2). It was found that cells in the G₁ phase were stimulated by IL 2 which increased transferrin receptor concentration after a lag of a few hours. Subsequently, the cells entered the S phase and the receptor levels remained high throughout the S, G₂ and M phases of the cell cycle.

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The biosynthesis of the EGF receptor was examined in the epidermoid carcinoma cell line A431 and five novel cell lines from human squamous cell carcinomas possessing high numbers of EGF receptors. Newly synthesized EGF receptors were visualized by labeling with [³⁵S]methionine and immunoprecipitation with a monoclonal anti-EGF receptor antibody. In addition, the processing of the EGF receptor and its intracellular transport was analyzed by distinguishing cell surface receptors from intracellular receptors and by treating cells with inhibitors such as tunicamycin, monensin and brefeldin A. These analyses revealed that in all the tumor cell lines the EGF receptor is synthesized as a glycosylated protein of Mr 160, 000 which is converted to the receptor of Mr 170, 000 through posttranslational glycosylation. The receptors of Mr 160, 000 and 170, 000 appeared to possess high mannose type oligosaccharide chains because endoglycosidase H treatment reduced their molecular weights by approximately 30, 000. A431 was the only tumor cell line studied that secreted the truncated EGF receptor of Mr 110, 000. In A431 cells, the truncated EGF receptor was generated from a protein of Mr 60, 000 through tunicamycin-and monensin-sensitive glycosylation. A431 cells treated with monensin secreted the truncated receptor as a Mr 95, 000 form.

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Cycloheximide strongly antagonizes the induction of sisterchromatid exchanges by ethyl methanesulfonate or mitomycin C in human skin fibroblast and xeroderma pigmentosum cells (group A). Analogous behavior has been observed in several other species including Chinese hamster and plant cells. This report documents an exception to that pattern: cycloheximide fails to antagonize UV-induced sister chromatid exchange in xeroderma pigmentosum cells, whereas it does in normal human skin fibroblast cells. A genetic defect in these cells is postulated to alter the UV-mediated DNA recombination process.

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Ca²⁺-and phospholipid-dependent protein kinase (PKC) was activated by arachidonic and myristic acids. This activation by both fatty acids required the calcium ion. Acidic phospholipid was also required for the activation by myristic acid, while that by arachidonic acid was inhibited by phospholipid.

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The single-channel properties for monovalent and divalent cations of a voltage-independent cation channel from Tetrahymena cilia were studied in planar lipid bilayers. The single-channel conductance reached a maxi-mum value as the K⁺ concentration was increased in symmetrical solutions of K⁺. The concentration dependence of the conductance was approximated to a simple saturation curve (a single-ion channel model) with an apparent Michaelis constant of 16.3 mM and a maximum conductance of 354 pS. Divalent cations (Ca²⁺, Ba²⁺, Sr²⁺, and Mg²⁺) also permeated this channel. The sequence of permeability determined by zero current potentials at high ionic concentrations was Ba²⁺ ?? K⁺ ?? Sr²⁺>Mg²⁺>Ca²⁺.
Single-channel conductances for Ca2+ were nearly constant (13. 9 pS-20. 5 pS) in the concentrations between 0. 5 mM and 50mM Ca-gluconate. In the ex-periments with mixed solutions of K⁺ and Ca²⁺, a maximum conductance of Ca²⁺ (γ^Ca_max) and an apparent Michaelis constant of Ca²⁺ (K^Ca_m) were obtained by assuming a simple competitive relation between the cations. γ^Ca_max and K^Ca_m were 14. 0 pS and 0. 160 mM, respectively. Single-channel conductances in mixed solu-tions were well-fitted to this competitive model supporting that this cation chan-nel behaves as a single-ion channel. This channel had relatively high-affinity Ca²⁺-binding sites.

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Glycolipid compositions of three mouse myeloid leukemia cell clones, two that are sensitive to differentiation inducers (M1-T22 and M 1-S1) and one that is differentiation-resistant (M1-R1), have been compared. The T22 and S1 clones contained glucosylceramide (GlcCer), lactosylceramide (LacCer) and gangliotriaosylceramide (Gg₃Cer) as the major neutral glycolipids. The differentiation resistant clone, R1, was characterized by the appearance of globotriaosylceramide (Gb₃Cer) and a decrease of Gg₃Cer. There was a distinct difference in the ganglioside profile between the differentiation-inducible and-resistant clones: T22 and S1 cells contained no detectable amounts of ganglioside, whereas six different gangliosides were detected in the R1 clone. These gangliosides were isolated and identified as GM₃, GM₂, GM_1a, GD_1a, GMib, and a unique disialoganglioside, GD_1a, having the following structure:
Sialic acid α2 6
GalNAcβ1-4Ga1β1-1Cer.
Sialic acid α2-3Gal 3
Based on these comparative studies, the relationship between the glycolipid composition and the differentiation potential of leukemia cells is discussed.

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The distribution of membrane sterols in the acrosome reaction of Japanese abalone sperm was studied by freeze-fracture techniques using a polyene antibiotic, filipin, as a probe for membrane sterols. In the unreacted sperm, the plasma membrane covering the acrosome showed a random distribu-tion of filipin-sterol complexes, while in the outer acrosomal membrane, filipin-sterol complexes were rarely observed. In the initial stage of the acrosome reac-tion, filipin-sterol complexes were found densely distributed in the plasma mem-brane throughout the acrosomal region. On the subjacent outer acrosomal mem-brane, filipin-sterol complexes were densely packed in the apical area, cor-responding to the truncated cone region. This apical area consistently showed such regionalization of filipin-sterol complexes during the acrosome reaction. As the acrosomal process elongated, the intramembrane particle (IMP)-free areas which appeared in the acrosomal process membrane were found to be displaced by densely distributed filipin-sterol complexes. These results indicate a possible regionalization of membrane sterols related to the acrosome reaction.

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33 kDa protein of neutrophil is a lipocortin-like protein, as proposed from its biochemical properties, amino acid composition, and the homology of its amino acid sequence to human lipocortin I. The localization and translocation of 33 kDa protein (p33) in blood cells of guinea pig were studied by immunoblotting (Western blotting) and immunocytochemical fluorescence methods using polyclonal and monoclonal mouse anti-p33 an-tibodies. The protein was determined to be present only in the cytoplasm of neutrophils, but not in cells such as the monocyte, lymphocyte, platelet, and other bone marrow cells. The translocation of the protein from cytoplasm to cell membrane was coupled with the increase in intracellular calcium ion and in superoxide generation induced by a chemotactic factor. These findings suggest that p33 may have an important role not only in the regulatory mechanism of phospholipase A₂ (PLA₂) activity but also in other transmembrane signaling.

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Quiescent 3Y1 cells in monolayer cultures were dispersed with trypsin-EDTA, suspended in various media, and the cellular heat production was measured in a flow-type microcalorimeter set at 37°C. A linear relationship was found to exist between the number of cells applied to the microcalorimeter and the heat output. Increasing concentrations of bovine serum albumin (BSA) and of fetal calf serum (FCS) added in Dulbecco's modified Eagle's medium (DEM) enhanced the heat output to the same saturation level. Trypsin inhibitor added in DEM enhanced the heat output, but to a lower saturation level than FCS or BSA did, indicating that BSA has an activity to enhance cellular heat production by a mechanism other than neutralizing residual trypsin. The heat output was found to gradually decrease in the microcalorimeter. This reduction was not enhanced by a two-fold dilution of the medium (DEM plus FCS) with phosphate-buffered saline, indicating that this reduction is not caused by the depletion of nutrients and serum factors in the medium. Similarly, when cells were incubated for 155 or 220 min in suspension in DEM plus BSA at 37°C and applied to the microcalorimeter, the heat output decreased. However, no signifi-cant reduction of the heat output was observed after holding the cells at 0°C in suspension for the same period. This and other facts suggest that depletion of O₂ dissolved in the medium is involved in the gradual decrease in heat output. The present results will be especially useful in a future investigation in which we plan to measure the heat production in cells of a monolayer culture by flow microcalorimetry in an attempt to determine one of the parameters of the cellular physiological state.

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