We studied the relationship between the force and velocity of microtubule sliding in demembranated sperm flagella of the sea urchin, Hemicentrotus pulcherrimus, under auxotonic conditions following a quick release of the tension between sliding microtubules. The shape of the force-velocity curve was independent of the concentration of Mg-ATP over the range of 3.7 to 350 μM and appeared either linear or was the reverse of the hyperbolic curve seen for muscle. The power, calculated as the product of velocity and force, passed through a peak at c. 0.7 F_max (the maximal isometric force). Thus, the maximal power is attained at a larger relative load than in muscle. The sliding velocity at 0.1 F_max showed a hyperbolic dependence on Mg-ATP concentration, with a K_m of 210 μM and a V_max of 19 μm•sec^-1. The maximal force did not significantly change over the Mg-ATP concentration range of 3.7 to 350 μM. These results are discussed in terms of a crossbridge model similar to the one originally proposed by Huxley. It is suggested that the dynein crossbridge cycle is characterized by a relatively rapid rate of attachment and a relatively slow rate of detachment.

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Based on the observation that photoautotrophically grown Chlorella cells fail to complete cell division under anaerobic condition in the dark, we devised a heterotrophic synchronous culture (HSC) system for this green alga. The system consists, at a temperature of 21°C, of a 32±1 h period under 3% CO₂ in air and the successive 12±1 h period under 0.5%O2±99.5% N₂ (the sum of the two periods is equal to 44 ± 1 h), using a semi-balanced medium containing glucose in an inorganic salt solution. This procedure could be successively repeated several times and the division index was practically 100%, as it is for Chlorella in the photoautotrophic synchronous culture (PSC). We believe this is the first successful HSC achieved by the method of alternating atmospheric oxygen tension.
Synthetic patterns for DNA, RNA and protein as well as chlorophyll during the cell cycle of Chlorella in HSC were similar to those observed in PSC, and the respiratory activity of actively growing cells in HSC was twice that of those in PSC. It is hoped that this system, under the regimen of high and low oxygen tension, can be utilized for other eukaryotic cells.

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Vitronectin is a 75 kilodalton (kDa) cell-adhesive glycoprotein found in animal blood and connective tissue, also termed serum spreading factor, S-protein, and epibolin. It promotes attachment and spreading of animal cells on tissue culture dishes, and it also binds to collagen. We established four mouse hybridoma lines producing monoclonal antibodies (M1, M2, M4 and M5) to human vitronectin. By immunoblotting, both epitopes recognized by M4 and M5 were suggested to exist in the amino terminal 5 kDa portion of vitronectin, and both Ml and M2 bound to the adjacent 35 kDa portion. Cell spreading on vitronectin-coated dishes was inhibited by M4 = M5 > M1, but not by M2. Collagen binding to vitronectinwas inhibited by M2 >M4 =M5, but not by Ml. These results indicate that the collagen-binding site is located near the cell-binding site in the amino terminalhalf of vitronectin. Independent inhibition of vitronectin binding to the cell and to collagen by these monoclonal antibodies will provide a potential tool to dissect the structure and function of vitronectin.

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Nerve growth factor (NGF) induced the activities of acetylcholinesterase (AChE) and Na⁺, K⁺-ATPase concomitant with neurite outgrowth in PC12h cells, while dibutyryl cyclic AMP (DBcAMP) caused the induction of AChE activity and neurite outgrowth but not Na⁺, K⁺-ATPase activity. A nonproteinaceous extract isolated from the inflamed skin of rabbits inoculated with vaccinia virus (Neurotropin) induced neurite outgrowth and cell surface change similar to NGF without affecting AChE activity. The results suggest that NGF, DBcAMP and Neurotropin act on PC12h cells through different mechanisms.

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The structure of RNA on the surface of Ehrlich ascites tumor cells was studied under an electron microscope using both plasma polymerization replica films and ultrathin sections of the cells. Necklace-like structures were found on the cell surface where anti-RNA antibody was bound in replica film, and particles which resemble cytoplasmic ribosomes in size and density were found distributed sparsely on the cell surface in ultrathin sections. These particles were found to gather at one pole of the cell surface after the cell was incubated at 4°C with anti-RNA antibody and then incubated at 37°C for 10 min in antibody-free medium. On the other hand, L1210 cells which do not bind with anti-RNA antibody showed hardly any such structures on the cell surface. These results suggest that RNA on the surface of Ehrlich ascites tumor cells is present in the form of particles.

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Early cellular events with respect to protein synthesis and the steady-state level of cellular myc (c-myc) mRNA were analyzed in the tumor necrosis factor (TNF)-sensitive human promyelocytic leukemia cell line HL-60 and in its TNF-resistant variant HL-60R after their exposure to TNF. Addition of TNF at 100 units (U)/ml induced de novo synthesis of two proteins with apparent molecular masses of 100 kDa and 40 kDa in HL-60 cells. The induced synthesis of the 100 kDa protein continued for 6 h, while that of the 40 kDa protein was transient. The 100 kDa protein was detectable in HL-60R cells which were maintained in medium containing 1, 000 U/ml TNF, whereas the synthesis of the 40 kDa protein could be transiently induced by TNF at 10⁵ U/ml. Dot blot hybridization revealed that the steady-state level of c-myc mRNA in HL-60 cells was transiently reduced by TNF at 100 U/ml but remained at a reduced level for 6 h when 105 U/ml TNF was present. In HL-60R cells, TNF at 105 U/ml could transiently reduce the c-myc mRNA level. These results showed that induction of the synthesis of a 40 kDa protein and a reduction in the steady-state level of c-myc mRNA were concomitant with cellular sensitivity to the cytostatic action of TNF in HL-60 cells.

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To examine the roles of the cytoplasms of differentiated somatic cells on nuclear gene expression, reconstituted cells (RC-cells) were isolated clonally by fusing karyoplasts (isolated nuclei) from neomycinresistant mouse teratocarcinoma PCC4-neo^r cells with cytoplasts (isolated cytoplasms) of chloramphenicol (CAP)-resistant rat myoblasts L₆TG.CAP^r cells, and after double selection in the medium containing 400 μg/ml of neomycin and 100 μg/ml of CAP (G418 plus CAP medium). The RC-cells were characterized by the presence of two genetic markers, neomycin-and CAP-resistance, by the absence of latex beads which had incorporated into karyoplast donor PCC4-neo^r cells as a cytoplasmic physical marker, and by the similar karyotypes as that of parental PCC4-neo^r cells.
In contrast to the teratocarcinoma cybrids previously isolated, all the isolated RC-clones expressed myoblast-like morphologies of three types. The phenotypic expression of these RC-cells was compared with that of PCD-1 cells, a teratocarcinoma-derived myoblast line. RC-cells and PCD-1 cells did not express alkaline phosphatase (ALPase) activity while parental PCC4-neor expressed it strongly. After induction of myogenic differentiation by treatments with excess thymidine and conditioned medium, two clones were capable of forming short multinucleated cells. The protein synthetic patterns of RC-cells analysed by two-dimensional polyacrylamide gel were different from PCC4-neo^r cells, and quite resembled those of PCD-1 cells. Particularly, multinucleated RC-clones expressed α-tropomyosin, and contained 10 nm filaments, characteristic markers of early myogenic cells.
These results suggest that the RC-cells are myoblast-like cells, that a few of them maturate to partially differentiated myogenic cells, that the rat myoblast cytoplasm contains regulatory factor(s) able to determine the myogenic cell lineage of the undifferentiated stem cells, and that this factor is continuously expressed in these myoblasts.

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The purpose of the present study is to analyze the effect of serum or chick embryo extract (CEE) on the neuronal differentiation of the mouse neural crest cells. When the crest cells were cultured in the medium containing serum at low concentration (5% calf serum), neurite outgrowth was observed. The active outgrowth was detected at 3-4 days in culture. However, in the medium supplemented with 20% calf serum, no neurite appeared, and the crest cells remained fibroblast-like. The differentiation of adrenergic neurons was observed when the crest cells were cultured in the medium containing CEE along with serum.

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