Abstract: Adult rat hepatocytes seeded in a noncoated plastic dish containing serum-free medium formed a monolayer within 24 h of culture. Those seeded in a dish coated with a proteoglycan fraction isolated from rat liver reticulin fibers attached to the dish but did not spread within 4 h, and then gradually assembled to form floating spherical aggregates (spheroids) with a diameter of 120 +/- 40 micron, within 72 h. The proteoglycan fraction appeared to contain dermatan sulfate, heparan sulfate and an unidentified glycosaminoglycan in its glycan moieties by glycosaminoglycan analysis after pronase digestion and high molecular weight proteoglycan molecules (mw: over 300,000 and about 200,000) by SDS-PAGE analysis. Cells seeded in dishes coated with these defined glycosaminoglycans and heparin assembled to form hemispheroids and multilayer islands, but not floating spheroids, within 72 h of culture. Dermatan sulfate had a stronger ability to induce hemispheroids than heparan sulfate or heparin. As the hemispheroid and multilayer islands were the intermediate form between monolayer and floating spheroids, the glycosaminoglycan moieties of the proteoglycan fraction were thought to participate in the formation of spheroid.

Abstract: Vitronectin is a cell-adhesive glycoprotein in serum and plasma, also termed serum spreading factor and complement S-protein. It consists of a mixture of a polypeptide of molecular weight 75 kilodalton (kDa) and its nicked product of 65 kDa plus 10 kDa. By a quantitative immunoblotting assay, human blood samples could be classified into three distinct vitronectin types; type I (58% of the population) was 75 kDa rich and 65 kDa poor, type II (35% of the population) contained approximately equal amounts of 75 kDa and 65 kDa, and type III (5% of the population) was 75 kDa poor and 65 kDa rich. The vitronectin type did not correlate with age, sex, or ABO blood type.

Abstract: The biosynthesis of the EGF receptor was examined in the epidermoid carcinoma cell line A431 and five novel cell lines from human squamous cell carcinomas possessing high numbers of EGF receptors. Newly synthesized EGF receptors were visualized by labeling with [35S]methionine and immunoprecipitation with a monoclonal anti-EGF receptor antibody. In addition, the processing of the EGF receptor and its intracellular transport was analyzed by distinguishing cell surface receptors from intracellular receptors and by treating cells with inhibitors such as tunicamycin, monensin and brefeldin A. These analyses revealed that in all the tumor cell lines the EGF receptor is synthesized as a glycosylated protein of Mr 160, 000 which is converted to the receptor of Mr 170, 000 through posttranslational glycosylation. The receptors of Mr 160, 000 and 170, 000 appeared to possess high mannose type oligosaccharide chains because endoglycosidase H treatment reduced their molecular weights by approximately 30, 000. A431 was the only tumor cell line studied that secreted the truncated EGF receptor of Mr 110, 000. In A431 cells, the truncated EGF receptor was generated from a protein of Mr 60, 000 through tunicamycin-and monensin-sensitive glycosylation. A431 cells treated with monensin secreted the truncated receptor as a Mr 95, 000 form.

Abstract: Ca2+- and phospholipid-dependent protein kinase (PKC) was activated by arachidonic and myristic acids. This activation by both fatty acids required the calcium ion. Acidic phospholipid was also required for the activation by myristic acid, while that by arachidonic acid was inhibited by phospholipid.

Abstract: Hybridomas secreting monoclonal antibodies to transferrin receptor (TFR) were isolated. One of these antibodies, U-1, recognized the cytoplasmic domain of TFR and the others, N-2 and W-3, recognized its cell surface domains. Only antibody W-3 competed with transferrin (TF) for binding to TFR. Antibody U-1 bound to purified TFR but not to 35S-or 125I-TFR in cell extracts. 125I-Antibody U-1 bound to TFR alone in cell extracts when TFR was bound to antibody N-2-Sepharose 4B, but even in the presense of cell extracts it did not bind to TFR bound to antibody W-3-Sepharose 4B. Antibody W-3 co-precipitated TFR and a protein of about 30 kDa from cell extracts, and also reacted with the 30 kDa protein in cell extracts in the absence of TFR. Based on these results, the existence of two different states of the cytoplasmic domain of TFR is discussed.

Abstract: We used monoclonal antibodies specific for acetylated and nonacetylated alpha-tubulin to detect and to localize microtubules containing acetylated alpha-tubulin (stable microtubules) in the pathogenic protozoa Tritrichomonas foetus and Trichomonas vaginalis. SDS-PAGE analysis showed that tubulin is a major protein of both parasites, being enriched in cytoskeletal preparations of whole cells extracted with Triton X-100. The monoclonal antibodies, which recognize all isoforms of alpha-tubulin (B-5-1-2) and only acetylated alpha-tubulin (6-11B-1), bind to the tubulin of T. foetus and T. vaginalis as seen by immunoblotting. Tubulin-containing structures were localized using immunofluorescence microscopy and transmission electron microscopy of the whole cytoskeleton previously incubated in the presence of the anti-tubulin antibodies and a second antibody-gold complex, and then processed using the negative staining or replica techniques. The results obtained indicate that, in addition to the flagellar microtubules, those which form the peltar-axostyle system represent stable microtubules containing acetylated alpha-tubulin.

Abstract: An epithelial cell line, designated as SGE1, has been established from isolated rat renal glomeruli. SGE1 cells are able to grow continuously in a serum-free medium at similar growth rates to those in the medium containing serum. Quantitative studies of the cells in the serum-free condition demonstrated that SGE1 cells required a collagen type I or IV, fibronectin, or laminin-coated substratum for adhesion and growth, and among them, collagen type I and IV were most effective. Essential medium supplements for the adhesion and growth were epidermal growth factor and transferrin, respectively, and both effects were noticeably enhanced with linoleic acid. Morphological observation found that in a monolayer culture, SGE1 cells formed domes, and in a collagen embedding culture, they formed cystic spheres having features of a simple cuboidal epithelium, polarized formation of microvilli and tight junctions as well as a lateral cell membrane with cytoplasmic projections. In addition, SGE1 cells expressed Fx1A antigens, which are nephritogenic antigens on their microvilli.

Abstract: Two expression plasmids (pSVIFN gamma/BPV97 and pSVIFN gamma/AdDHFR) for constitutive production of human interferon-gamma (HuIFN-gamma) were constructed and introduced into the two different mammalian cell lines, mouse C127 cells and Chinese hamster ovary (CHO) cells. Genetically engineered C127 and CHO cells grew on microcarriers having high productivity of HuIFN-gammas for at least six months. Isoelectric focusing patterns and molecular weight analyses suggest that C127- and CHO-HuIFN-gammas are glycoproteins and that both HuIFN-gammas have different molecular structures. The development of a microcarrier culture system for genetically engineered mammalian cells has enabled us to prepare glycosylated HuIFN-gammas on a large scale.

Abstract: The single-channel properties for monovalent and divalent cations of a voltage-independent cation channel from Tetrahymena cilia were studied in planar lipid bilayers. The single-channel conductance reached a maximum value as the K+ concentration was increased in symmetrical solutions of K+. The concentration dependence of the conductance was approximated to a simple saturation curve (a single-ion channel model) with an apparent Michaelis constant of 16.3 mM and a maximum conductance of 354 pS. Divalent cations (Ca2+, Ba2+, Sr2+, and Mg2+) also permeated this channel. The sequence of permeability determined by zero current potentials at high ionic concentrations was Ba2+ greater than or equal to K+ greater than or equal to Sr2+ greater than Mg2+ greater than Ca2+. Single-channel conductances for Ca2+ were nearly constant (13.9 pS-20.5 pS) in the concentrations between 0.5 mM and 50 mM Ca-gluconate. In the experiments with mixed solutions of K+ and Ca2+, a maximum conductance of Ca2+ (gamma Camax) and an apparent Michaelis constant of Ca2+ (K Cam) were obtained by assuming a simple competitive relation between the cations. Gamma Camax and K Cam were 14.0 pS and 0.160 mM, respectively. Single-channel conductances in mixed solutions were well-fitted to this competitive model supporting that this cation channel behaves as a single-ion channel. This channel had relatively high-affinity Ca2+-binding sites.

Abstract: Human recombinant tumor necrosis factor (TNF) stimulated the growth of confluent human fibroblasts (FS-4) in the presence of fetal calf serum. Epidermal growth factor (EGF) similarly stimulated cellular growth; however other mitogenic factors such as insulin, fibroblast growth factor, 12-0-tetradecanoyl-phorbol-12-acetate and Ca2+ ionophore A23187 did not. The growth-stimulating action of TNF was not synergistic with the activity of EGF in the presence of serum. TNF induced a rapid increase in the binding of transferrin to the cell surface, followed by a return to the basal level within 5 min. A similar increase in transferrin binding was observed in FS-4 cells ex-posed to EGF. In contrast, insulin caused a prolonged stimulation of transferrin binding. These results suggest that TNF and EGF generate similar or identical intracellular signals for cellular growth and the regulation of transferrin receptor expression.

Abstract: We report here on a structural association of single cilia, via their striated rootlets, with the Golgi complex in epithelial cells and stromal fibroblasts of rabbit ciliary processes of the eye. The structure is designated a Golgi-cilium complex and its likely role in aqueous humor production is discussed.

Abstract: 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.

Abstract: Human diploid fibroblasts (TIG-3) were shown to attach and spread onto substrata coated with collagen, fibronectin, laminin and vitronec-tin. The cell attachment to these proteins required divalent cations. Mg2+ stimulated the cell attachment to all the proteins, while Ca2+ alone was not effec-tive for the attachment to collagen and laminin. A mild trypsin treatment had prevented cells from attaching to the laminin, while it had no effect on the at-tachment to the other proteins. The fibronectin fragment, which retained cell binding activity, inhibited the cells from attaching and spreading onto fibronec-tin, but it did not cause any inhibition on the other proteins. The synthetic pep-tide GRGDSP inhibited the cells from attaching and spreading onto fibronectin and vitronectin, while it did not cause any inhibition on collagen and laminin. In attempts to isolate distinct receptors for these proteins, we were able to purify proteins very similar to the fibronectin and vitronectin receptors of human placenta. Based on the differential properties of the attachment of TIG-3 cells to these proteins and biochemical data, we indicate that human diploid fibroblasts have distinctive binding sites (receptors) for collagen, fibronectin, laminin and vitronectin.

Abstract: We have previously described some of the characteristics of an actin binding protein, 53 K protein, purified from porcine brains. The purification procedure was revised in order to investigate of this actin binding protein further. A Scatchard plot analysis showed that the association constant between actin and the 53 K protein has around the same value as those reported for the fascin-actin and for the filamin-actin interactions. The binding experiments also demonstrated the occurrence of competitive binding with other actin binding proteins such as filamin, alpha-actinin, caldesmon and tropomyosin for the actin filament. Antibody was produced against brain 53 K protein and further purified on an affinity column. Immunoblot analysis using the antibody showed that this protein is localized in both the soluble and membraneous fraction of the brain. Other tissues such as liver and lung also contain 53 K protein. The immunoblot analysis also revealed that the gelation product of rat brain extract described by Palmer et al. contains immunoreactive polypeptides having slightly lower molecular weights and more basic isoelectric points than porcine brain 53 K protein. Immunological localization of the 53 K protein within HeLa and BS-C-1 cells showed that this protein is distributed throughout the cell in small granules, and in some regions of the cell, these granules were aggregated into much larger granules.

Abstract: 33 kDa protein of neutrophil is a lipocortin-like protein, as proposed from its biochemical properties, amino acid composition, and the homology of its amino acid sequence to human lipocortin I. The localization and translocation of 33 kDa protein (p33) in blood cells of guinea pig were studied by immunoblotting (Western blotting) and immunocytochemical fluorescence methods using polyclonal and monoclonal mouse anti-p33 an-tibodies. The protein was determined to be present only in the cytoplasm of neutrophils, but not in cells such as the monocyte, lymphocyte, platelet, and other bone marrow cells. The translocation of the protein from cytoplasm to cell membrane was coupled with the increase in intracellular calcium ion and in superoxide generation induced by a chemotactic factor. These findings suggest that p33 may have an important role not only in the regulatory mechanism of phospholipase A2 (PLA2) activity but also in other transmembrane signaling.

Abstract: Two methods of qualitative analysis of sequence distribution in DNA and protein are presented. The first method is based on the finding that the frequency of occurrence of each nucleotide in a defined sequence with functional significance more or less deviates from uniform distribution. The deviation found in this defined sequence seems to parallel the function of this sequence. In the second method, two model compounds (trypsin and its inhibitor) have been used to see the topological fit between their local structures. Acrophilicity parameter for amino acid was used to construct the topological structure. Both methods may find practical application in algorithms to design functional DNA and protein molecules.

Abstract: 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 neomycin-resistant mouse teratocarcinoma PCC4-neor cells with cytoplasts (isolated cytoplasms) of chloramphenicol (CAP)-resistant rat myoblasts L6TG.CAPr cells, and after double selection in the medium containing 400 micrograms/ml of neomycin and 100 micrograms/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-neor cells as a cytoplasmic physical marker, and by the similar karyotypes as that of parental PCC4-neor 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-neor cells, and quite resembled those of PCD-1 cells. Particularly, multinucleated RC-clones expressed alpha-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.

Abstract: Several cell lines growing in protein- and lipid-free synthetic medium secreted cell-adhesive protein(s) into the medium. The conditioned medium (CM) of one of these cell lines, mouse L•P3, showed the highest cell at-tachment-promoting activity (CPA) among them. Cell-adhesive protein(s) in the CM of L•P3 cells (L•P3-CM) were separated into two types by sequential affinity column chromatography employing gelatin-Sepharose 4B and heparin-Sepharose 4B. One was a gelatin- and heparin-binding cell-adhesive protein (GCP), and was identified as a cellular form of mouse fibronectin. The other was a gelatin-non-binding and heparin-binding cell-adhesive protein (GNCP). The CPA of GNCP preparation was effective for the cell-attachment and spreading of both epithelial and fibroblastic cells. The CPA of GNCP prepara-tion was not blocked by the antiserum and scarcely inhibited in the presence of the synthetic cell attachment-promoting peptide Gly-Arg-Gly-Asp-Ser-Pro, a competitive inhibitor of fibronectin. This suggests that the structure of the cell-attachment site of GNCP is different from that of fibronectin. The GNCP preparation showed little cross-reactivity with anti-mouse laminin antiserum in enzyme-linked immunosorbent assay (ELISA). These results demonstrate the possibility that GNCP in L•P3-CM is a novel cell-adhesive protein distinct from fibronectin or laminin. The secretion of the two types of cell-adhesive pro-teins by L•P3 cells is discussed.

Abstract: Glycolipid compositions of three mouse myeloid leukemia cell clones, two that are sensitive to differentiation inducers (M1-T22 and M1-S1) and one that is differentiation-resistant (M1-R1), have been compared. The T22 and S1 clones contained glucosylceramide (GlcCer), lactosylceramide (LacCer) and gangliotriaosylceramide (Gg3Cer) as the major neutral glycolipids. The differentiation resistant clone, R1, was characterized by the appearance of globotriaosylceramide (Gb3Cer) and a decrease of Gg3Cer. There was a distinct difference in the ganglioside profile between the differentiation-inducible and -resistant clones: T22 and S1 cells contained no detectable amounts of ganglioside, whereas six different gangliosides were detected in the R1 clone. These gangliosides were isolated and identified as GM3, GM2, GM1a, GD1a, GM1b, and a unique disialoganglioside, GD1 alpha, having the following structure: (formula; see text) Based on these comparative studies, the relationship between the glycolipid composition and the differentiation potential of leukemia cells is discussed.

Abstract: Insulin-like growth factor-I (IGF-I) stimulated the phosphorylation of cytoskeletal 350-kDa and 300-kDa proteins which were immunoprecipitated with antibodies against brain high molecular weight microtubule-associated proteins in quiescent rat 3Y1 cells. The data on the effec-tive concentrations of IGF-I and 125I-labeled IGF-I binding indicated that type I IGF receptors mediate this IGF-I effect. Platelet-derived growth factor (PDGF) as well as phorbol ester (TPA) also stimulated the phosphorylation of these proteins. These proteins, whether immunoprecipitated from cells stimulated by insulin, IGF-I, TPA, PDGF, or epidermal growth factor, produced very similar phosphopeptide mapping patterns irrespective of the stimulant. The results suggest the possibility that these growth factors and phorbol esters may activate a common protein kinase which is responsible for the phosphorylation of the 350-kDa and 300-kDa proteins in cells.

Abstract: The mechanism through which nonmembranous lipid inclu-sion bodies consisting of cholesteryl esters accumulate in the cytoplasm was studied. Most lipid inclusion bodies in macrophages after 24 h incubation with anisotropic cholesteryl oleate liquid crystals were surrounded by a limiting mem-brane. The limiting membrane, however, could not be observed after further incubation for 48 h in the presence of esterastin, which is known to be an inhibitor of lipase and esterase. Under these conditions, the levels of hydrolysis and re-esterification of cholesteryl esters were less than 15% and 5% of the control ones, respectively.These results suggest that the inclusion bodies were transferred from lysosomes to the cytoplasm, with partial hydrolysis of cholesteryl esters, in addition to through the pathway via microsomes.

Abstract: A cell line (HuL-1) derived from normal fetal human liver was adapted to grow continuously in a modified Eagle's minimum essential medium without serum or hormones. The population doubling time of this adapted cell line (HuL-1-317) was about 72 h and the modal number of chromosomes was 54. The morphology of HuL-1-317 cells was round in the absence of serum, but at 37 degrees C with the addition of serum (1-10%), the cells flattened. HuL-1-317 cells had a low level of alkaline phosphatase activity. However the enzyme activity was slightly enhanced by the combination of prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and a hypertonic concentration of NaCl after 3 days of incubation at 37 degrees C. The increase in alkaline phosphatase activity with the four agents was further amplified dose-dependently by the pretreatment of the cells with serum. The stimulatory effect of the serum was evident at concentrations as low as 1%, and was maximal at 20%. The half life of the effect of serum on alkaline phosphatase induction was 48 h at 37 degrees C. Serum alone could not enhance the enzyme activity without the four agents. The present results indicate that serum contributes to the regulation of alkaline phosphatase induction by the combination of prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCl in fetal human liver cells (HuL-1-317).

Abstract: 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 degrees 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.

Abstract: Upon tryptic digestion of synaptosomes, ATPase activities decreased in the order of Cl(-)-ATPase greater than or equal to Na+,K+-ATPase greater than anion-insensitive Mg2+-ATPase. Upon synaptosome treatment with hypotonic solution, Cl(-)-ATPase or anion-insensitive Mg2+-ATPase was slightly inactivated, while Na+,K+-ATPase underwent a much larger degree of inactivation. ATP-Mg inhibited the ATPase digestion in the hypotonic-solution-treated synaptosomes in a concentration-dependent manner, but not in the untreated synaptosomes. These results suggest that trypsin-digestible site of Cl(-)-ATPase are present on both sides of the synaptosomal plasma membrane, and the ATP-Mg binding site of the enzyme is located on the inner surface of the membrane.

Abstract: We have studied protein acylation in neutrophils of guinea pigs using [3H]myristate. A large number of neutrophil proteins were acylated with exogenously added myristic acid. The myristoylation was detected on 110, 77, 56, 54, 52, 42, and 37 kDa proteins. These myristoylations were stronger in peripheral blood than in peritoneal cells. Myristic acid was found to be covalently linked by an amid bond to these proteins since the proteins were resistant to boiling, chloroform/methanol and hydroxylamine treatment. Most myristoylated proteins appeared to be associated with the membrane fraction, while some of the proteins such as 77 kDa one was distributed also in the cytoplasm and translocated from the cytoplasm to the plasma membrane by stimulation. Lysozyme was myristoylated in vitro by the N-hydroxysuccinimide ester of myristic acid. The myristoylated lysozyme had an ability to be associated with phospholipid liposomes, and the membrane-associated lysozyme became a substrate of the rat brain Ca2+- and phospholipid dependent protein kinase (protein kinase C). These results indicate that myristoylation in neutrophil proteins may have an important role in metabolic regulation through their membrane association.

Abstract: 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.

Abstract: The effects of exogenously added glycosphingolipids on the differentiation of mouse myeloid leukemia cells (M1-T22) have been studied. Eight gangliosides and ten neutral glycosphingolipids were tested in terms of their induction of phagocytic activities on the leukemia cells. N-Acetyl-neuraminosyllactosylceramide (NAc-GM3) was the most effective glycolipid for inducing the activity. By the addition of 25 micrograms/ml of NAc-GM3, about 70 percent of the cells acquired phagocytic activity within 20 h incubation. GM1a showed about half the activity of the GM3. In the case of the neutral glycosphingolipids, lactosylceramide (CDH) and globotriaosylceramide (CTH) showed significant effects on the induction of phagocytic activity. Preincubation of the cells with the NAc-GM3 enhanced the effect of dexamethasone as a differentiation inducer on M1-T22 cells. When a human promyelocytic leukemia cell line, HL-60, was preincubated with the NAc-GM3 ganglioside, induction of the phagocytic activity, together with inhibition of the cell growth by phorbol ester (TPA), were markedly enhanced. From these observations, the NAc-GM3 ganglioside seems to act as a modulator of differentiation of mouse myeloid leukemia cells and also of HL-60 cells.

Abstract: Distribution and temporal change of free calcium concentration ([Ca2+]i) in single guinea pig gastric chief cells were visualized by a digital imaging microscope equipped with a microspectrofluorometer. The distribution was not homogeneous; a higher [Ca2+]i area was often localized in some restricted regions of the endoplasm and also at the peripheral cytoplasm just beneath the plasma membrane. When stimulated with cholecystokinin, [Ca2+1]i increased transiently in the apical peripheral cytoplasm and in the endoplasmic regions. This Ca2+ mobilization which precedes the biphasic pepsinogen secretion was composed of a rapid Ca2+ release from the intracellular store(s) as well as a rapid and a more sustained Ca2+ entry from the extracellular space.

Abstract: The 15 kDa protein is the most abundant low molecular weight Ca2+-binding protein, different from calmodulin, in eggs of sea urchin, Hemicentrotus pulcherrimus (9). The data from the amino acid sequence demonstrated that the 15 kDa protein belonged to the troponin C superfamily. Based on immunofluorescent and immunomicroscopic observations, we showed that the 15 kDa protein localized in the nuclei of fertilized eggs and mitotic apparatus of dividing eggs. Microinjection of the antibody against 15 kDa protein into sea urchin blastomeres resulted in the arresting of cell division. These results suggest that the 15 kDa protein plays an important role in mitosis of sea urchin egg.