A voltage-dependent and Ca²⁺-activated cation channel recent-ly found in the vacuolar membrane of the yeast Saccharomyces cerevisiae was incorporated into planar lipid bilayers and further characterized in macroscopic and single channel levels. Single channel conductances for various cations were in the order: NH₄⁺ > K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, and were nearly consistent with the order of permeability ratio estimated from reversal potentials determin-ed by macroscopic measurement. Up to 6 mM of Ca²⁺ added to the cis (cytoplasmic) side opened the channel, but higher concentrations closed the channel without affecting the single channel conductance. Ba²⁺ closed the chan-nel without opening. Further, large anions such as gluconate closed thechannel from the cis side. In addition to the above channel, a small cation-selective chan-nel of about 40 pS was found.

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We have demonstrated simultaneous measurement of the membrane potential and cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) by utilizing a dual-laser flow cytometer in embryonic rat hippocampal cell suspensions. Veratrine, a Na⁺ channel activator, induced both membrane depolarization and elevation of [Ca²⁺]_i. These actions of veratrine were all reversed by the presence of tetrodotoxin (TTX). These findings suggest that Na⁺ channels are functionally expressed in the cells and the activation of Na⁺ channels increases [Ca²⁺]_i. The usefulness of the flow cytometric analysis in elucidating the expression of membrane functions in the embryonic central nervous systems (CNS) is discussed.

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The secretion of tissue-type plasminogen activator (t-PA) from melanoma cells (Bowes) was investigated with or without monensin treatment. Monensin inhibited secretion of t-PA from the cells to the medium in a dose-and time-dependent manner. The inhibition was accompanied by an intracellular accumulation of t-PA. Electrophoretic enzymography of the cell homogenate showed the main lytic zone at 72 kDa, which reacted with the IgG of anti-t-PA. Analysis of the cell organelles using ultracentrifugation with a discontinuous sucrose density gradient revealed that the activity and the antigen of t-PA were observed near the discontinuous phase of the sucrose gradient. Analysis of ³H-mannose-and ³⁵S-methionine-labeled t-PA in the cell organelles revealed that the radioactivity of each was increased by monensin treatment, and that such treatment increased the ratio of ³H-mannose-related glycoprotein to ³⁵S-methionine-related protein. The sugar chain of intracellular t-PA was analyzed with endoglycosidase H and N-glycanase, which reduced the molecular weight of t-PA by 4.5-10 kDa, indicating the intracellular presence of a high-mannose type sugar chain and a complex-type sugar chain of t-PA. t-PA secreted from the monensin-treated cells possesses a high-mannose type sugar chain only. Therefore, monensin alters the secretion of t-PA by abnormal glycosylation.

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The effect of a tumor-promoting phorbol ester on spreading behavior was investigated to clarify the involvement of the interactions between cells and substratum in the maintenance of cell viability and the control of cell proliferation. BSC-1 cells did not spread and lost cell viability after a 24-h in-cubation in the absence of calf serum. Addition of calf serum initially induced radial spreading and then polarized spreading, with the formation on stress fibers and focal contact-like structure, and enhanced survival. Vitronectin also induced both radial spreading and polarized spreading, and enhanced cell sur-vival. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced radial spreading with actin ribbons in the absence of serum. It improved the survival of cells at-tached to the substratum, but not in suspension. TPA suppressed polarized spreading, formation of stress fibers and of focal contact-like structure, and cell proliferation, in the presence of serum. Phorbol did not have any effect. These results suggest that enhancement of radial spreading and inhibition of polarized spreading of BSC-1 cells by TPA are closely related to the enhancement of cell survival and inhibition of cell growth.

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Calcium ionophore, A23187, induced a large depolarization of the membrane (referred to as the activation potential) of sea urchin Pseudocen-trotus depressus eggs, which was very similar to the fertilization potential induc-ed by a sperm. The egg membrane abruptly overshot to +12 mV, then slightly fell, and again slowly polarized to about +10 mV, where the membrane sustain-ed for about 10 sec. Peak values of this second component were found to be very sensitive to the external Na⁺ concentration and external K⁺ concentration. These features were very similar to the second component (component B) of the fertilization potential. On the other hand, even when removed Ca²⁺ from exter-nal artificial sea water, the activation potential was found to show essentially the same time course and magnitude as those in Ca²⁺ -containing artificial sea water. Thus the second component of the activation potential elicited by A23187 is probably induced by the increment of Na⁺ and K⁺ permeabilities, which might be triggered by the release of Ca²⁺ from the intracellular storing sites. These results suggest that component B of the fertilization potential is caused by the elevation of internal Ca²⁺ concentration.

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Some evidences have been found that virulency in paramyxoviruses depends on the sensitivity of the cleavage recognition site ofthe F glycoprotein to serine type proteases. In this report, the interactionenergies between the active site of trypsin and the cleavage recognition sites inparamyxoviruses are calculated. Results show that van der Waals energy andelectrostatic energy contribute to the sensitivity. The virulencies of some myxo-and retro-viruses are then predicted on the basis of the two calculated interac-tion energy values.

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Analysis of the amino acid sequence in protein (deviation analysis) suggests that the binding between trypsin and the enveloped virus is the first step of their interaction, which occurs in a specified configuration. It is possible that the distance between their active sites is important for the viral sen-sitivity to trypsin, which is related to the virulency of the enveloped virus.

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A new endothelial cell growth factor (f-ECGF) was partially purified from the cultured medium of human fibroblast cells of embryonic lungs. The partially purified f-ECGF induced neovascularization in rabbit cor-nea. It showed a selective growth stimulatory activity on the endothelial cells in vitro, whereas acidic-and basic-fibroblast growth factors (a-and b-FGFs) show-ed a broad spectrum of growth stimulation among tissues or cells. f-ECGF did not compete with the binding of a-FGF to the cell surface receptor in HEP-G2 hepatoblastoma cell lines. These results indicated that f-ECGF is a new en-dothelial cell growth factor distinct from a-and b-FGFs which are known to be potent endothelial cell growth factors.

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Flagellar axonemes that lack the outer dynein arms and a part of the inner dynein arms were prepared by extracting demembranated axonemes of an outer-arm less mutant of Chlamydomonas with a solution containing 0.3 M KC1 and 10 μM ATP. When reactivated with 1 mM ATP, the extracted axonemes, whose beat frequency before the extraction had been 26.9±2.6 Hz (26°C, n=10), beat with a reduced frequency of 14.9±3.7 Hz (26°C, n=10). The decrease in beat frequency depended on the KC1 concentration and on the presence of ATP in the extracting solution. The bend angle and the curvature of bend were not affected by the extraction. Electron microscopic observation showed that 28.5% of the inner arms were weak in appearance or lost in the extracted axoneme. Electrophoresis of the axonemal proteins indicated that one of the high molecular weight polypeptides of the inner dynein arm was reduced in quantity. These results suggest that some part of the inner arms has the function of increasing the flagellar beat frequency but is not essential for flagellar motility.

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Spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, were demembranated with solutions containing various detergents and then reactivated with ATP. The reactivation rates of sperm demembranated with 0.05% (w/v) saponin, Brij 58, or CHAPS (3-[(3-cholamidopropyl) dimethylam-monio]-1-propanesulfonate) were less than 10%; increasing the concentration of detergent up to 0.1% (w/v) did not improve the reactivation rate. High reac-tivation rates were obtained with 0.04% (w/v) Triton X-100, 0.05% Nonidet P-40, 0.04% CHAPS + 0.01% Triton X-100, or 0.04% CHAPS +0.01% Nonidet P-40. The flagellar waveform as well as the relationship between the beat frequency and the ATP concentration did not vary among these four preparations. Unlike the live sperm, Triton-extracted sperm failed to undergo rotation of the flagellar beat plane for more than one and a half revolutions when lateral vibration was imposed on the head in a plane that rotated around the head axis. However, the sperm demembranated with a solution containing 0.04% CHAPS and 0.01% Nonidet P-40 responded to the imposed rotatory vibration by rotating their flagellar beat plane through more than 10 cycles. Elec-tron microscopic studies showed that the membrane were completely removed from around the axoneme by treatment with Triton X-100 or CHAPS + Nonidet P-40. These results indicate that the rotation of the beat plane does not require the presence of the plasma membrane, and also that the mechanism con-trolling the beat plane is independent of that controlling other parameters of flagellar movement.

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By employing in gel competitive reassociation, which distinguishes two DNA preparations, to clone anonymous DNA fragments with altered primary structure, we isolated a clone (BL-1) from a rat DNA which gave an extra, apparently altered, DNA band in a specific tissue (brain) when wused it aS a probe for Southern hybridization. The sequence of BL-1 was very similar to a portion of LINE, a highly repetitive sequence. Southern hybridiza-tion analysis with oligonucleotide probes representing a portion of the BL-1 sequence confirmed the presence of the brain-specific band. Another, different band specific to HindIII digests of heart DNA was also detected with the same oligonuleotide probes. Furthermore, we found that poly (A) +RNA hybridized with BL-1 was enriched in brain. These results suggest that there are tissue specific alteration of genomic DNA in rats which may be associated with transcriptional activation specific to the sequence. Alternative explanations of the results are also discussed.Hiroshi Yokota

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