The change in distribution of centrosomal phosphoproteins was examined in sea urchin eggs from fertilization to the first cleavage by immunofluorescence staining with the anti-phosphoprotein antibodies, MPM-1 and MPM-2. The antibodies reacted with female pronuclei in un fertilized eggs as well as centriolar complexes located at the base of sperm flagella. After insemination, male and female pronuclei fused together to form a zygotic nucleus which was visualized by staining of fertilized eggs with the antiphosphoprotein antibodies. No major change in staining pattern was detected in extracted whole eggs until mitosis. As the fertilized eggs approached mitosis, however, the antigens started to redistribute from nuclei to the perinuclear position where the mitotic centrosomes were located. Detailed immunofluorescence observation of isolated spindles revealed that the phosphoantigens were retained in isolated structures. A major 225 kd polypeptide was recognized by the antibodies, suggesting that the 225 kd protein is a phosphocomponent of centrosomes. The area recognized by the antibody in mitotic poles enlarged with the progress of mitosis, suggesting that the antigens were apparently localized in the centrosphere. Centrospheres prepared from isolated spindles by salt extraction strongly reacted with the antibodies. One or two bright dots, which may represent centrioles, were visible in the isolated centrosphere. At the end of mitosis, the antigens again appeared in the newly formed daughter nuclei. Centriole-containing cytasters and centriole-free monasters were parthenogenetically induced in un fertilized eggs (Kuriyama and Borisy, (1983) J. Cell Sci. 61: 175-189). The antibodies stained centers of both the asters whether they contained centrioles or not, indicating that the antibodies recognizes the components of the pericentriolar material.

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Using full length cDNA introduced into COS-7 cells, two species of P-450 with entirely different physiological functions have been expressed in enzymatically active form. One is P-450d, which is known to reside in the microsomes of rat hepatocytes where it acts as a drug-metabolizing enzyme; the other is P-450(SCC), which catalyzes the conversion of cholesterol to pregnenolone in the rate-limiting reaction of steroidogenesis in mitochondria of adrenal cortex cells. Northern blot and immunoblot analyses revealed that the mRNA and protein of these P-450 species were efficiently produced in COS-7cells. The protein contents amounted to nearly 0.1% of the total cell protein as estimated from immunoblotting and low temperature CO difference spectra. The subcellular localization of the products indicated that they were correctly sorted to the microsomes and mitochondria, respectively. We have succeeded in eliciting most of the activity of the expressed microsomal P-450d by reconstruction with NADPH-cytochrome P-450 reductase, while the optimal conditions for the mitochondrial enzyme in the COS cells remain to be studied.
These results show the applicability of the COS-7 expression system to investigations of the functions of members of the P-450 superfamily whose cDNA has been newly isolated.

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The morphological effects of Brefeldin A (BFA) on the parotid acinar cells of a rat were investigated at the stage of active resynthesis of secretory materials following administration of the secretogogue, isoproterenol. Incubation with BFA resulted in: a) marked dilation of the rough endoplasmic reticulum (RER), b) involution of the Golgi complex to rudimentary forms which disseminated throughout the cytoplasm, and c) agenesis of secretion granules. It appears that the primary action of BFA is inhibition of the export of secretory materials from the RER toward the Golgi complexes. Histochemical staining indicated the thiamine pyrophosphatase (TPFase) positive saccules of the Golgi stack to undergo degradation in autophagic vacuoles. In contrast, small vesicles showing the osmium reducing activity characteristic of cis elements, including osmium negative vesicles, continued to be present throughout a 4-h period of investigation, indicating the cis and, most likely, medial elements to be the components of the rudimentary Golgi complexes. On removal of the drug, a large number of transport vesicles appeared immediately from the RER and carried secretory materials to the rudimentary Golgi complex, so that the organdies were rapidly reconstructed within 30-60 min, followed by the reaccumulation of secretory granules by 90 min. It is thus indicated that the size and configuration of the Golgi complex is regulated by a dynamic equilibrium of the transport of secretory materials, and that the rudimentary Golgi complex containing cis and probably medial elements may function as the smallest units of the Golgi complex for full development as seen under normal conditions.

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A temperature-sensitive cell-cycle mutant, tsJT16, which has been isolated from Fischer rat fibroblasts, was defective in the function(s) that operated soon after growth stimulation. When G₀-arrested tsJT16 was stimulated to proliferate, it entered the S phase after 12-15 h at 34°C but failed to do so at 40°C. The function mutated in tsJT16 was required to be normal for the first 4 h or less for cells to transit from the G₀ to S phase. The induction of cell-cycle-dependent genes such as c-fos, c-myc and ornithine decarboxylase was observed at both temperatures after growth stimulation. Although an increase in total protein synthesis occurred at both temperatures after growth stimulation, synthesis of one protein (p70) (pI 7.8 and Mr 70, 000) was inhibited at 40°C. Synthesis of p70 was negligible in G₀-arrested cells and blocked by actinomycin D in serum-stimulated cells at 34°C. These results suggest that tsJT16 has a ts defect in one of the signal transduction processes to induce gene activation. tsJT16 was also defective in progression of the G₁ phase of growing cells, consistent with the previous results in which growth stimuli were required at G₁ for continuation of proliferation.

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tsJT16 is a temperature-sensitive (ts) mutant of rat fibroblasts that has a ts defect in a function operating soon after the growth stimulation from the G₀ phase. After the growth stimulation, the cells express several cell-cycle-dependent genes at both temperatures while they fail at the nonpermissive temperature to synthesize a protein p70 identified on two-dimensional gel electrophoresis. Here we report that 1) synthesis of p70 began within 1 h of stimulation, continued up to the 7th hour and then decreased; 2) the half-life of p70 was shortened after 6 h after the stimulation; 3) p70 was localized in the nuclear fraction; 4) p70 was likely to be a primarily induced protein; 5) mRNA of p70 was supposed to be synthesized exclusively within 2 h of growth stimulation. These and the previous results suggest that p70 is a nuclear protein responsible for the early stage of transition of cells from the G₀ toward the S phase and is induced via a different signal transduction sequence from that for the c-fos gene.

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The effects of the hydrophobic peptide antibiotics leucinostatins A and B, originally isolated by Arai, T., Y. Mikami, K. Fukushima, T. Utsumi, and K. Yazawa. (J. Antibiotics (1973) 26: 157-161), on the functions of rat liver mitochondria were examined. At a concentration of 240 nM, these compounds completely inhibited state 3 respiration and ATPase activity that was stimulated by weakly acidic uncouplers. However, at higher concentrations, they induced uncoupling, probably by their protonophoric action. The uncoupling action was potentiated by known phosphoryl transfer inhibitors such as venturicidin, DCCD and oligomycin. The binding site of leucinostatins at lower concentrations was suggested to be located at, or very close to that of venturicidin. The potencies of the two analogues of leucinostatin were almost the same for all their actions. Their effects were very similar to those of the peptide antibiotics A20668's, which have been used as leucinostatins without any chemical and biological confirmation that they are in fact leucinostatins. Thus, the chemical structures of leucinostatins are thought to be analogues to those of the A20668's.

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We isolated and cultured erythroblastic islands (EI) from the spleens of phlebotomized mice using a combination of collagenase digestion, unit gravity sedimentation, and Percoll density gradients separation. The isolated El were composed of surrounding erythroid cells and central stromal macrophages (Mφ), which were identified by Forssman antigen. While 60% of the erythroblasts incorporated bromodeoxyuridine, the Mφ did not. EI could be maintained on a plastic dish for a short period in the presence of erythropoietin.
Two hours later, the central Mφ spread well and bound to erythroblasts via cytoplasmic processes. One day later, erythropoietic activity on the Mφ surface continued, although their processes had retracted. Some EI showed synchronized expansion of erythroblasts and others showed differentiation to reticulocytes. Two days later, about 50% of the EI still showed erythropoietic activity and most erythroblasts differentiated to the orthochromatic stage. On the other hand, the Mφ secreted colony-stimulating activity during the culture. It was infrequently observed that erythroid and myeloid populations simultaneously expanded on a central Mφ. These results indicate that this El culture system is useful for studying interactions between the stomal Mφ and hematopoietic cells.

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Two types of DNA ligase, I and II, have been purified approximately 4, 000-fold from mouse testes and 500-fold from nuclei of mouse spermatocytes. DNA ligase I and II consisted of single polypeptides with molecular weights of 95, 000 and 65, 000, respectively, according to the estimation by SDS-polyacrylamide gel electrophoresis and the AMP-binding assay. Ligase activities were higher in premeiotic spermatogonia and spermatocytes than those in liver and bone marrow cells. Moreover, DNA ligase II showed rapid increase during meiotic prophase and a decrease in round spermatids. Since this behavior of DNA ligase II is consistent with that of m-rec and DNA polymerase β, both of which have been shown to be involved in DNA recombination in meiotic cells, DNA ligase II might be an enzyme which works at the final step of meiotic recombination reaction.

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