The stiffness (flexural rigidity) of live sperm flagella, Triton-demembranated flagella (axonernes), trypsin-digested axonemes, and doublet microtubules of the axonemes in echinoderms was determined from the relationship between their deformation when a stream of medium was applied and the viscous resistance of the medium acting on the flagellum. The stiffness of the flagellum beating in seawater was 5.8×10^-21 Nm² for bending in the direction perpendicular to the beating plane and 4.2×10^-22 Nm² for bending within the beating plane. A similar difference in stiffness from the difference in bending directions was found in reactivated flagella with 1 Mm ATP. The stiffness of live flagella immobilized in CO₂-saturated seawater and axonemes in ATP-free medium was similar to that of beating flagella for bending in the direction perpendicular to the beating plane. The stiffness of motionless flagella significantly decreased with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and vanadate. The trypsin-digestion of motionless axonemes did not change their stiffness. The stiffness of doublet microtubules was 1.4 ×10^-23 Nm² in 0.1 mM ATP medium and 6.1×10^-23 Nm² in ATP-free medium. These results suggest that doublet pairs lying parallel to the beating plane of the flagellum retain fewer cross-bridges than doublet pairs lying perpendicular to the beating palne.

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The relationship between intracellular period modulation and external environment change was investigated from the viewpoint of internal information coding in Physarum plasmodium. For the external conditions, concentration changes of attractant (galactose) and repellent (KC1) were used, and the internal responses were measured as the thickness oscillation of the plasmodium. (i) Period of the intracellular oscillation decreased when the concentration of attractant was increased and when the concentration of repellent was decreased, (ii) The period increased when the attractant was decreased and when the repellent was increased, (iii) The larger concentration change induced the larger period modulation, (iv) These responses were observed when the change of concentration was greater than a threshold value. From these results, it was clarified that the relative change in environmental condition is encoded on the relative period modulation in intracellular oscillation. This means that the period change does not directly represent the environment itself but represents the change of its condition. Thus, it is further suggested that the plasmodium estimates the environmental condition based on the relationship between the previous external condition and the present one.

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To study the effect of a bovine brain sialoglycopeptide (SGP) on the nerve cell growth, PC12 cells were used as a model system. Though the inhibition of cell growth, measured by the 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT) assay, by SGP was slight under the serum concentration of 10%, it was clearly detected under that of 1 to 2%. For the preparation of SGP, filtration through a nitrocellulose membrane was useful.

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The modulation of phosphoinositidase C (PIC) beta activity upon interferon treatment in Burkitt lymphoma cells (Daudi) and its localization and expression have been analyzed by Western blotting, immunocytochemical and immunoelectronmicroscopy analysis. Results have disclosed an early increase of phosphatidyl-inositol-bisphosphate (PIP₂) hydrolysis at nuclear level upon interferon (IFN) treatment paralleled by the evidence of an increase of PIC beta 1 expression. PIC beta 1 expression has been detected in the nuclear compartment also in a clone of Daudi cells selected for the resistance to the antiproliferative action of interferon alpha but no modulation of the enzyme has been detected upon interferon treatment. Since no changes in terms of PIP₂ hydrolysis have been found at nuclear level in this selected line, we suggest that the antiproliferative action of interferon on Burkitt lymphoma cells is mediated by a possible recruitment of nuclear PIC beta 1 expression.

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A procedure is described for changing the medium surrounding individual cells attached to the bottom of a cell chamber. A small hole at the "apex" of a plastic U-tube allowed application and withdrawal of medium. The medium to be applied was per fused through the U-tube by pressure at one end and suction at the other. To prevent premature delivery of new medium from the U-tube, suction of the outlet dominated resulting in a net withdrawal of medium from the cell chamber. The flow of medium through the hole could be reversed rapidly by arresting the suction with an electromechanical valve. In this way it was possible to obtain 95% replacement of medium with in 60 ms. A pressure transient arising from the closure of the valve was damped by the presence of a small air bubble in the system. To secure a precise deposition of medium and minimize the risk of mechanical disturbances to the cell it was essential to be able to inspect the medium changes visually. For this purpose the fluorescent indicator rhodamine B bound to dextran proved satisfactory. Free rhodamine B could not be used because it had biological effects, as was evident from studying ATP-regulated K⁺ channels in pancreatic β-cells. When using a purpose-designed syringe pump for per fusing the U-tube, the technique allows well controlled exposure of individual cells to test substances added together with dextran-linked rhodamine B.

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Demecolcine (Colcemid), an inhibitor of spindle fiber formation in M phase, induced apoptosis in V79 cells. At a concentration of 0.01μg/ml demecolcine, V79 cells proliferated exponentially as well as controls, although temporal M phase accumulation occurred 6 h after the addition of demecolcine. At 0.1 μg/ml, the cells became hyperploid after remaining in the M phase for some time. Apoptosis occurred in V79 cells exposed to demecolcine at a concentration of 0.03μg/ml. Apoptosis was defined as the appearance of a sub-G1 peak in DNA histograms and a ladder pattern of fragmented DNA in gelelectrophoresis.

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The epidermal growth factor (EGF)-induced endocytosis of its receptor is an obligatory pathway for the cellular regulation of the EGF-specific receptor (EGF-R). BNER4 is a mouse Balb/3T3 cell line transfected with human EGF-R complementary DNA (CDNA). B4/src-13 and B4/src-24 are BNER4 cells transfected with a viral oncogene v-src. Indirect immunofluorescence study demonstrated that EGF-R was mostly localized at the perinuclear region in BNER4cells at 60 min after EGF addition, whereas it was diffusely distributed throughout the cytoplasm in its v-src transfectants. Double indirect immunofluorescence study further confirmed that EGF-R was localized in lysosomes in BNER4 and B4/src-13 cells at 60 min after EGF addition. Intracellular distribution of the Golgi apparatus, clathrin-coated vesicles and early endosomes were similar in all cell lines. However, the lysosomes detected by anti-lysosomal membrane protein (LGP85) antibodies were diffusely distributed throughout the cytoplasm in the v-src transfectacts. By contrast, in the parental BNER4 cells, the lysosomes were mostly localized in the perinuclear region. The organization of microtubules, but not of actin, was markedly different between BNER4 cells and its v-src transfectants. Nocodazole, which depolymerizes microtubules, altered the distribution of the lysosomes and EGF-R in BNER4 cells. Both intracellular lysosome distribution and microtubule organization in nocodazole-treated BNER4 cells were found to be similar to those in its v-src transfectants without nocodazole treatment. These findings support the notion that changes in lysosome distribution may be correlated with microtubule reorganization by v-src in mouse Balb/3T3 cells.

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