A cytolysin, theta-toxin, preferentially binds to membrane cholesterol surrounded by phospholipids with 18-carbon hydrocarbon chains in cholesterol-rich region.

We have previously suggested the existence of two distinctive states of cholesterol in erythrocyte and lymphoma cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens [Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Ando, S., & Nagai, Y. (1988) Eur. J. Biochem. 176, 95-101; Ohno-Iwashita, Y., Iwamoto, M., Ando, S., Mitsui, K., & Iwashita, S. (1990) Biochim. Biophys. Acta 1023, 441-448]. To understand factor(s) which determine membrane cholesterol heterogeneity, we analyzed toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (phosphatidylcholine/phosphatidylglycerol = 82:18, mol/mol). Liposomes containing phospholipids with 18-carbon hydrocarbon chains at both positions 1 and 2 of the glycerol have both high- and low-affinity toxin-binding sites with Kd values similar to those of intact erythrocytes, whereas liposomes with hydrocarbon chains containing 16 or fewer carbons at either position 1 or 2 have only low-affinity toxin-binding sites. The cholesterol/phospholipid ratio, in addition to the length of phospholipid hydrocarbon chain, also determines the number of toxin-binding sites, indicating that at least these two factors determine the topology of membrane cholesterol by creating distinctively different affinity sites for the toxin. Since theta-toxin binding detects specific populations of membrane cholesterol that are not detectable by the measurements of susceptibility to cholesterol oxidase and cholesterol desorption from membranes, the toxin could provide a unique probe for studying the organization of cholesterol in membranes.     

A case of renin producing leiomyosarcoma originating in the lung.

A 54-year-old woman was referred to our hospital for the treatment of a tumor of the right chest wall. Clinical examination revealed hypertension, hypokalemia, metabolic alkalosis, hyperaldosteronism and hyperreninemia. Computed tomography and an abdominal echogram indicated a tumor in the right phrenic area and two tumors in the retroperitoneum near the pancreas head. After the surgical resection of these tumors, the primary reninism was diminished. The pathological diagnosis of these tumors was leiomyosarcoma. Plasma active and inactive (trypsin-activated) renin activities (PRA) were 85.7 and 38.9 ng angiotensin I/ml/h, respectively. These PRA did not respond to either postural stimulation or suppression by the volume expansion. Active and inactive renin activities in a right phrenic area tumor were 208 and 32 ng angiotensin I/mg protein /h, respectively. Those of an abdominal tumor were 196 and 30 ng angiotensin I/mg protein/h, respectively. Renin mRNA identical in molecular size to that of the human kidney was identified by northern blot analysis. This is the first case report of renin producing leiomyosarcoma derived from the lung, which is characterized by relatively lower plasma prorenin concentrations.     

Secretion of endothelin-1 in human endothelial cell line but not in B cell line by transfection of preproendothelin-1 cDNA.

Stable transformants with preproendothelin-1 (preproET-1) cDNA were established for the study of the regulation of endothelin-1 (ET-1) biosynthesis in human cells. ET-1, a potent vasoconstrictor peptide, is produced by endothelial cells and is secreted into the blood at a low level. Human preproET-1 cDNA was introduced into two immortal human cell lines, t-HUE2, an endothelial cell line, and Raji, a B cell line, with Ecogpt selection. Several stable transformants of t-HUE2 expressed extraordinarily high levels of preproET-1 specific mRNA and secreted ET-1 into serum-free culture medium, while the transformants of Raji cells expressed high levels of ET-1 mRNA, but secreted a negligible amount of ET-1. Immunocytochemical studies of intracellular ET-1 content revealed that there were some defects in the translation or processing of preproET-1 in the B cell line transformants. In addition, the ratio of ET-1 to ET-1 precursor (big ET-1) was much higher in the t-HUE2 transformants than in normal endothelial cells, suggesting that t-HUE2 transformants (for example t-HUE2-1) possess high levels of endothelin converting enzyme (ECE). The establishment of stable transformants producing high levels of ET-1 in serum-free medium will be useful for the study of cell-type-specific translation and processing to mature ET-1, and of the regulatory factors of ECE.     

The B subunit of cholera toxin enhances DNA synthesis in rat hepatocytes induced by insulin and epidermal growth factor.

The B subunit of cholera toxin, which binds to ganglioside GM1, enhanced DNA synthesis in rat hepatocytes in primary culture induced by insulin and/or epidermal growth factor. The effect was dose-dependent, and whole cholera toxin, activating adenylate cyclase, showed a higher effect than the B subunit alone. The B subunit acted additively with other agents that also increase cyclic AMP levels. A competitive antagonist of cyclic AMP could not suppress the effect of the B subunit completely. These data suggest that the effect is independent of the cyclic AMP signal pathway, and that GM1 plays a role in hepatocyte proliferation.     

Down-regulation of prostaglandin E2 receptors in regenerating rat liver and its physiological significance.

The properties of prostaglandin (PG) E2 receptors in regenerating liver were studied using rat hepatocytes in primary culture. The control cells possessed stereo-specific PGE2 receptors with Bmax and Kd values, at 4 degrees C, of 526 fmol/mg protein and 6.5 nM respectively. In cells from regenerating liver after 70% hepatectomy, Bmax was reduced to 42-43% that of the controls; Kd did not change. Administration of indomethacin before surgery prevented Bmax reduction. These results indicate that PGE2, produced during the regeneration process, evoked cellular events and regulated the density of its receptors.     

Structure and biosynthesis of the xylose-containing carbohydrate moiety of rice alpha-amylase

Suspension-cultured cells of rice secrete alpha-amylase into the culture medium. It has been shown that the mature form of the alpha-amylase contains xylose-bearing N-linked oligosaccharide: (formula; see text) We demonstrate that suspension-cultured cells of rice secrete alpha-amylase containing oligomannose-type oligosaccharides in the presence of 1-deoxymannojirimycin or tris(hydroxymethyl)aminomethane. On the other hand, alpha-amylase purified from germinated rice seedlings contains several kinds of oligomannose-type and N-acetyllactosamine-type oligosaccharides. The processing pathway of oligosaccharide moieties in rice cells is discussed on the basis of a comparison of these oligosaccharides structures.     

Cell surface changes accompanying aging in human diploid fibroblasts : III. Division age and senescence revealed by concanavalin A-mediated red blood cell adsorption

The relationship between cell size, [³H]thymidine incorporation capacity, and cell surface property of human diploid fibroblasts was investigated using the concanavalin A (ConA)-mediated red blood cell (RBC) adsorption assay. Small cells in late passage populations adsorbed RBCs well with the RBC coating method (in which ConA-coated RBCs are adsorbed to fibroblasts) as did large cells of this population, while small cells in early passage populations did not. The RBC adsorption capacity of rapidly dividing cells with this method differed among young, middle-aged and old cell populations. The results suggest that temporal cell size and [³H]thymidine incorporating capacity is not a measure of the division age of human diploid cells at the individual cell level. On the other hand, RBC adsorption with the fibroblast coating method (in which RBCs are adsorbed to ConA-coated fibroblasts) occurred to non-dividing cells of the populations. Thus, the increase in RBC adsorption with this method is considered to be a reflection of the increase in non-dividing cells at phase III. Our results support the hypothesis that RBC adsorption with the RBC and fibroblast-coating methods represents a cell surface marker for division age and senescence of human diploid cells, respectively, at the individual cell level.     

Hormonal requirements for the larval-pupal transformation of the epidermis of Manduca sexta in vitro

In the tobacco hornworm, Manduca sexta, metamorphosis occurs in response to two releases of ecdysone that occur 2 days apart. Epidermis was explanted from feeding final-instar larvae before the first release of ecdysone and was cultured in Grace's medium. When exposed to 1 μg/ml of β-ecdysone for 24 hr and then to hormone-free medium for 24 hr, followed by 5 μg/ml of β-ecdysone for 4 days, the epidermis produced tanned pupal cuticle in vitro. During the first 24 hr of exposure to β-ecdysone, the epidermis first changed its cellular commitment to that for pupal cuticle formation (ET₅₀ = 14 hr), then later (by 22 hr) it became committed to tan that cuticle. Then, for most of the pupal cuticle to be tanned, at least a 12-hr period of culture in hormone-free medium was required before the cuticle synthesis was initiated. Consequently, some events prerequisite to sclerotization of pupal cuticle not only occur during the ecdysone-induced change in commitment but also during the ecdysone-free period. When the tissue was preincubated in 3 μg/ml of juvenile hormone (JH I or a mimic epoxygeranylsesamole) for 3 hr and then exposed to both ecdysone and juvenile hormone for 24 hr, it subsequently formed larval cuticle. The optimal conditions for this larval cuticle formation were exposure to 5 μg/ml of β-ecdysone in the presence of 3 μg/ml of epoxygeranylsesamole for 48 hr. When the epidermis was cultured in Grace's medium for 3 days and then exposed to 5 μg/ml of β-ecdysone for 4 days, 70% of the pieces formed pupal cuticle. By contrast, if both ecdysone and JH were added, 77% formed larval cuticle. Therefore, the change from larval to pupal commitment of the epidermal cells requires not only the absence of JH, but also exposure to ecdysone.     

Isolation,Structure and Physiological Activities of Piericidin B,Natural Insecticide Produced by a Streptomyces

Piericidin B was isolated from mycellia of Streptomyces mobaraensis besides piericidin A. On the basis of IR, NMR and mass spectral studies together with chemical evidences, its structure was assigned as Id. Its physiological activities are also deescribd.