Mouse spleen lymphocytes require 2-mercaptoethanol for max-imal mitogenic activation in vitro. Previous studies indicate that the lym-phocytes are defective in the cystine transport activity and that they require 2-mercaptoethanolto utilize cystine. 2-Mercaptoethanol catalytically carries cys-teine moiety into the cells in a mixed disulfide form. Because cysteine is easily ox-idized to cystine in the culture medium, it has been not easy to precisely examine the effect of near-physiological concentrations of cysteine on the activation of lymphocytes. By controlling the cysteine content in the medium, we have review-ed the effect of cysteine to see if cysteine replaces 2-mercaptoethanol in enhanc-ing the DNA synthesis of lipopolysaccharide-stimulated lymphocytes. It was found that cysteine was less effective than 2-mercaptoethanol, and that cysteine fully replaced 2-mercaptoethanol when a selenium compound was sup-plemented. The effects of cysteine and selenium compounds were apparently in-dependent and additive. Among the selenium compounds examined, sodium selenite and L-selenocystine were much more effective in stimulating DNA syn-thesis than sodium selenate and L-selenomethionine.

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An acylpeptide called K-26, which was isolated from a culture filtrate of Bacillus sp. 503, inhibited motility of both live sea urchin sperm as well as Triton X-100-extracted sperm model. In each case, complete inhibition was observed at concentrations of more than 1 μM or 6 μM, respectively. This inhibitory effect of K-26 on the Triton-extracted sperm could not be reversed by reducing the concentration of K-26 in the reactivation medium containing 1 mM ATP. However, it was reversed when the ATP concentration in the reac-tivation medium was simultaneously reduced.
At concentrations below 4 μM, K-26 did not completely suppress the flagellar movement of the Triton-extracted sperm, but reduced the beat frequency in a competitive manner; the maximal beat frequency (Fmax) was 31.2 Hz whether in the absence or in the presence of 4 μM K-26, while Km for ATP was 0.19 mM or 0.28 mM, respectively. ATPase activity of isolated axonemes was increased severalfold in the presence of K-26. The sliding movement between the outer doublet microtubules in trypsin-treated axonemes was normal at K-26 concen-trations as high as 20 μM.
From these results, it was suggested that K-26 affects the mechanism that con-verts the sliding movement between the outer doublet microtubules into a ben-ding motion, and thereby inhibits the motility of the Triton-extracted sperm model.

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An acylpeptide called K-26 inhibited the motility of Triton-ex-tracted sea urchin sperm. This inhibition was canceled by the addition of both cAMP and a Triton X-100-soluble fraction of the sperm. The concentration of cAMP that was required to restore the motility was above 1μM. Incorporation of Pi into the Triton-extracted sperm was suppressed by K-26 at the same con-centration as that which inhibited the motility. A factor in the Triton-soluble fraction that was able to cancel the inhibition of the motility by K-26, was par-tially purified by hydroxylapatite and DEAE-cellulose column chromatogra-phies. In both of these steps, the activity to cancel the inhibition of the motility coeluted with that of cAMP-dependent protein kinase (cA kinase). The activity of cA kinase was also inhibited by K-26. The incorporation of Pi into the Triton-extracted sperm was suppressed in the presence of K-26 and was restored by the addition of both cAMP and the cA kinase fraction. From these results, it was suggested that K-26 affects the cAMP-dependent phosphorylation in the Triton-extracted sperm and thereby inhibits the process to convert the sliding of outer doublet microtubules into the bending of the flagellum.

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Analysis of mouse Swiss/3T3 fibroblasts and rat hepatoma H35 cells using the affinity cross-linking method revealed multiple forms of ¹²⁵I-insulin binding components (Mr >300, 000) in the absence of reducing agents. The same analysis, in the presence of reducing agents, revealed two major com-ponents (Mr=125, 000 and Mr=30, 000). The Mr=125, 000 component ap-peared to be the α-subunit of the high-affinity insulin receptor, whereas the small insulin-binding component of Mr=30, 000 was not a degradation product of the a-subunit but was apparently associated with the insulin receptor. We suggest that it is likely a novel component for regulating the function of insulin receptor.

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A cell line of retinoblastoma cells, TOTL-1, was established which hada very weakly amplified N-myc gene. It grew only in clumps that were hardly ever dispersed and showed retarded proliferation. Its doubling time was 160 h. TOTL-1 cells extended short thick processes in monolayer cultures on poly-D-lysine coated substratum, which were markedly different from those of Y79 cells. Light and electron microscopy observation of immunostaining with anti-pp60^srcsrc antibody revealed that the staining was accumulated in the polar regions of the cells.

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Spindles in anaphase of dividing primary spermatocytes of the grasshopper, Mongolotettix japonicus, were examined with a sensitive polarizing microscope combined with a time-lapse video recorder and a cinematographic apparatus. The pole-to-pole distance of the meiotic spindles was increased and the kinetochore fibers were more birefringent in the presence of 40 % D₂O. However, the rate of shortening of the kinetochore fibers in anaphase was not affected by D₂O. This indicates that D₂O did not inhibit microtubule disassembly in anaphase, supporting the earlier observations (3, 18) that D₂O did not "stabilize" the spindle microtubules at concentrations below 45 %. We confirmed that D₂O, at the concentration mentioned above, neither promotes nor inhibits the anaphase A. However, the overall sequence of anaphase was considerably extended in the presence of D₂O, presumably due to the increased pole-to-pole distance.

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Cultured fibroblasts of adult rats were used to determine whether they could take in retinol administered to the culture medium at physiological concentration. After the administration of retinol, cells were observed with a phase-contrast fluorescence light microscope (LM) and a transmission electron microscope (TEM). Retinol and retinyl fatty acyl esters (RFAE) stored in the cells were analyzed with high-performance liquid chromatography (HPLC). It was revealed that these fibroblasts could take in retinol in the medium at a concentration of 1 × 10^-7M and store it in lipid droplets in the cytoplasm as retinyl palmitate and other RFAE.

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Immunoblotting analysis demonstrated that the 260-kDa act-in-binding protein (260K protein) isolated from fertilized eggs of sea urchin, Hemicentrotus pulcherrimus, is immunologically different from mammalian fodrin (brain spectrin) and filamin. Rotary shadowing analysis revealed that the 260K protein is a flexible molecule measuring about 100 nm in length, which further indicated that it does not belong to the family of "spectrins". The isolated cortex was doubly stained with affinity-purified anti-260K protein antibody and rhodamine-labeled phalloidin to localize the 260K protein in comparison with the distribution of microfilaments. In unfertilized eggs and fertilized eggs at various developmental stages, the fluorescence for actin corresponded closely to the immunofluorescence for the 260K protein. In the unfertilized egg, the 260K protein showed a spotty distribution superimposed on the distribution of actin. The 260K protein was localized in the fertilization cone and the elongated microvilli which are composed of a microfilament bundle. A marked accumulation of the 260K protein was observed in the cleavage furrow. Immunoelectron microscopy using the secondary antibody labeled with colloidal gold particles indicated that the 260K protein is localized exclusively in the cell periphery beneath the plasma membrane. A high density of gold particles was detected along fibrous structures in many protrusions which seemed to correspond to microvilli. These observations suggested that the 260K protein is involved in the cortical cytoskeleton composed mainly of microfilament.

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