Resting platelets can be induced by epinephrine to shift to an activated state and to form aggregates, a basic event in haemostasis and thrombosis. Interaction of ¹⁴C-epinephrine with isolated membranes from human platelets was found to occur via a rapid, specific and reversible reaction which was maximal at 37°C over 60 min with a binding constant of 4.5-5.2 × 107 M-1 and 4-5 × 104 sites per platelet. Membranes become less sensitive to binding of epinephrine following their treatment with-SH group blockers, proteolytic enzymes, beta galactosidase and galactose oxidase. Binding was inhibited to varying degrees by alpha adrenergic agents and by mersalyl, a specific inhibitor of platelet ATPase activity of thrombosthenin, and to a much lesser extent by beta adrenergic agents at higher concentration. Detergent solubilized ¹⁴C-epinephrine labelled membranes electrophoresed on acryl-amide gels presented a pattern of labeling with two prominent peaks of radioac-tivity, suggesting that the label entered two components of apparent M.W. >200, 000 and 160, 000 daltons. These results indicate that high molecular weight components located on or within the platelet membrane recognize and bind epinephrine. One suggestion is that the complex phenomenon of platelet activation by epinephrine is initiated by events on the membrane involving an alpha adrenergic type effect of epinephrine binding by platelet membrane.

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The adhesive property was studied of Chang conjunctiva cells of human epithelial line. Cells dissociated with only EDTA aggregated in Ca²⁺-free and Mg²⁺-free conditions, whereas cells dissociated by trypsin did not aggregate under the same conditions. Cells dissociated with trypsin solution containing Ca²⁺ aggregated in the presence of Ca²⁺ and/or Mg²⁺. Cells dis-sociated by trypsin without Ca²⁺ showed less aggregation in the presence of divalent cations. In cases of cell adhesion to non-cellular substrates, the particular method of cell dissociation did not produce different results. Only a small fraction of inoculated cells attached onto plastic coated with bovine serum albumin or gelation. Many inoculated cells attached rapidly and spread onto plastic coated with collagen, when Mg²⁺ was present in the medium. These results were discussed with the hypothesis that Ca²⁺ and Mg²⁺ act differently in cell adhesion.

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The adhesive properties of normal BHK cells and of their transformed derivatives (pyBHK) were studied by examining the aggregation kinetics after different treatments for cell dispersion. Cells dispersed with 0.005 % trypsin with EDTA slowly aggregated only in medium with Ca²⁺. This aggregation was inhibited by cycloheximide. These cells acquired the ability to aggregate in medium without Ca²⁺ after preincubation in medium with Ca²⁺. Cells dispersed with lower trypsin concentrations (below 0.001 %) with EDTA aggregated rapidly in the absence, as well as in the presence of divalent cations. Cells prepared by the above two methods did not require Ca²⁺ for the adhesion process per se (Ca²⁺-independent adhesion), but did require this cation for recovery of adhesiveness damaged by the earlier trypsin treatment. Cells dispersed with 0.005 % trypsin solution containing Ca²⁺ aggregated rapidly in the medium with Ca²⁺, but did not aggregate in medium without Ca²⁺. The aggregation in Ca²⁺ was not inhibited by cycloheximide and required the continuous presence of Ca²⁺ in the assay medium. In this case, the mechanism must have required Ca²⁺ for adhesion per se (Ca²⁺-dependent adhesion). Both the Ca²⁺-independent and-dependent adhesion properties were found also in pyBHK cells, although the degree of Ca²⁺-independent aggregation was less in pyBHK than BHK cells.

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The relationship was analyzed between cell morphology and steroidogenesis of Y-1 cells to dibutyryl cyclic AMP in dose-related, as well as time sequence responses. Rounded cells were confirmed to be active in steroidogenesis. The time course study showed a bi-phasic pattern of steroid output, i.e., the first phase of 8 h showed a low secretion rate followed by a later accelerated phase for at least 24 h. In stimulant-free medium, most round-up cells showed a return to a flat and polygonal shape within 5 h of incubation. However, the elevated steroidogenic activity persisted for at least 7 h.

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Broom formation or broom-like aggregation of axopodia wasfound to be induced by treatment wth Con A (50 μg/ml) in the heliozoan, Echinosphaerium nucleofilum strain MA. This response was similar to capformation in lymphocytes. The broom formation was followed by axopodialdegradation and the subsequent cytolysis. At lower Con A concentrations (10to 30 μg/ml), broom formation was delayed considerably, while at higherCon A concentrations (90 to 125 μg/ml), axopodial degradation and cytolysisoccurred without broom formation. Pretreatment with 5×10_-3 M colchicine or2 μg cytochalasin B/ml modified the Con A-induced axopodial broom formation, which was termed here as windmill formation. Pretreatment with 10_-6 M PCMB or 10_-2 M NaN³ inhibited both broom and windmill formation. Pretreatment with 10_-4 M DNP produced no inhibitory effect on the Con A-induced axopodial rearrangement. The present data indicate that the ConA-induced rearrangement of heliozoan axopodia is closely related to cytoskeletal function.

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The synthesis and distribution of mitochondrial DNA (mt-DNA) were studied by light microscope autoradiography in the plasmodium of Physarum polycephalum. Mitochondria pre-labeled with ³H-thymidine (³H-TdR) for 3 h during early mitochondrial S phase (mS) and incubated in non-radioactive medium showed elongated shapes that developed to dumbbell-shaped, and the latter form of mitochondria divided. The incorporation of ³H-TdR into mitochondria occurred during mitochondrial growth but terminated before development to dumbbell-shapes, suggesting that they synthesized mtDNA during a limited period of mitochondrial division cycle. In addition, mitochondrial grains counts were reduced remarkably to about half during the late nuclear S phase, suggesting that Physarum mitochondria divided semi-synchronously during late nuclear S phase. The distribution of mtDNA pre-labeled during early or late mS was determined by the grain distribution of (a) labeling of both poles of dumbbell-shaped mitochondria, and (b) labeling of daughter mitochondria. The results indicated that mtDNA was distributed equally to daughter mitochondria, regardless of time of mtDNA replication.

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Photoelectric recordings showed that single isolated mouse myocardial cells cultured in vitro developed various types of arrhythmias, such as fluttering and fibrillatory beatings on addition of ouabain. Cell clusters also developed various types of arrhythmias on addition of ouabain. At low concen-trations of ouabain, the beating of cell clusters as a whole was synchronous and the rhythm of the synchronous beating was regular, but some component cells in the clusters showed rhythmic contractions accompanied with faint, fibrilla-tory beating of high frequency. At intermediate concentrations of ouabain, the beating of cell clusters as a whole was synchronous. But the rhythm of the synchronous beating was irregular. Many component cells in the clusters showed irregular, strong contractions accompanied with faint, fibrillatory beating of high frequency. The irregular, strong contractions of two cells 60 μm apart (4 cells apart) in the clusters were synchronous, but their faint, fibrillatory contractions were not. At high concentrations of ouabain, the cell clusters as a whole stopped beating, but many component cells in the clusters showed asynchronous fibrillatory beating of high frequency. Photoelectric recordings showed that the arrhythmias of component cells in cell clusters were essentially the same as those of single isolated myocardial cells. Ouabain-in-duced arrhythmias in both single isolated myocardial cells and in cell clusters disappeared on addition of quinidine sulfate. From these observations, the genesis and recovery from ouabain-induced arrhythmias of cell clusters are explained as essentially due to the genesis and recovery from arrhythmias of individual component cells of the clusters.

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Changes in intracellular concentrations of adenosine 3', 5'-monophosphate (cAMP) and guanosine 3', 5'-monophosphate (cGMP) were investigated in primary cultures of chick embryonic muscle cells grown in the presence of 5'-bromodeoxyuridine (BUdR). Cell fusion was completely in-hibited in BUdR-treated cultures; however, the effect was reversed when the BUdR-containing medium was replaced with normal medium. As in control cultures, a significant increase in the ratio of cAMP-to-cGMP was observed during the first few days in both the BUdR-treated and the BUdR-released cultures. The data indicate that cell fusion is not a necessary step for concentra-tion changes in cyclic nucleotides, which means that these changes might be acause but not an effect of myoblast fusion.

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Several spontaneous revertants able to grow in restrictive temperature (39°C) were isolated from a temperature-sensitive mutant (ts3) of murine leukemic cells L5178Y. The revertants showed reduced plating effi-ciencies and prolonged doubling times at 39°C when compared with wild type cells. The alanyl-tRNA synthetase activity was thermolabile in both ts3 cells and the revertants, indicating suppression as the mechanism of reversion. It was also demonstrated that glycerol protected the enzyme from thermal inac-tivation. The specific activities of alanyl-tRNA synthetase and alanine amino-transferase did not differ much in the wild type, mutant, and revertant cells. This indicates the unlikelihood of thermolabile enzyme overproduction or of increased intracellular concentration of L-alanine causing phenotypic suppres-sion in these revertants. The mechanism of sunnressinn is discussed.

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In the bovine heart, chondrification of the fibrous tissue occurs just after birth and proceeds to bony tissue. The ossification appears first in the right fibrous trigonum three months after birth and then in the left trigonum twelve months after birth. During ossification, the bone marrow structure appears to form a narrow space within the bony tissue and develops during the subsequent year. Light and electron microscopic examinations revealed development of typical hematopoiesis in the heart bone marrow. Hemoglobin was estimated in various erythroid cells by cytospectral analysis. The hematopoietic activity was retained for about 10 years in the heart bone.

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A simple method is described for in situ freeze-cleaving of fibroblasts grown on the Falcon plastic culture dish. The culture plate with cells is covered by another Falcon plastic plate previously coated with rat tail collagen so to sandwich the cells. The culture plate is then set on the freeze-etching apparatus. The freeze-etching apparatus used was the cold metal block type principally based on Bullivant and Ames' method. Fracturing was per-formed by removing the cover or the culture plastic plates. By setting the cul-ture plate below or above on the specimen stage, this method selectively exposed the EF and PF fracture faces of both the free surface and substrate contact surface of cultured cells.

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Cultured muscle cells treated successively with saponin at varied concentrations released protein initially at 2 x 10-5 g/ml saponin and then at 6 x 10-5 g/ml saponin. The first release was accompanied with creatine kinase activity, whereas the second release was not. Morphological observations indicated that the first protein leakage was derived mainly from myotubes and the second release mainly from mononucleated cells.

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Ehrlich ascites tumor-bearing mice were treated with colchi-cine or mitomycin C. Under colchicine, adenylate cyclase activity in tumor cells decreased to about a third of the untreated control level, whereas 5'-nucleotidase activity increased to almost twice the control level. Colchicine treatment resulted in about a 30 % reduction of ^3H-thymidine incorporation into DNA. Mitomycin C inhibited the incorporation of ³H-thymidine much more mark-edly but changes in the two enzyme activities were less than under colchicine. The two enzyme activities and ³H-thymidine incorporation into DNA showed parallel changes with lapse of time after inoculation of tumor cells into mice. Thus, the colchicine effects on the two enzyme activities seemed not due simply to inhibition of cell proliferation. The two enzymes seemed to be associated differently with the microtubular system in Ehrlich ascites tumor cells.

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