Development of the bitterling,Paracheilognathus himantegus (Cyprinidae), with a note on minute tubercles on the skin surface

The development of the eggs and larvae and minute tubercles on the skin surface ofParacheilognathus himantegus larvae were observed. The egg began to hatch approximately 68 hours after insemination and the larvae reached the free-swimming stage 23 days after hatching at water temperature of 22±1°C. The larval development and minute tubercles on the skin surface of this species were similar to those ofAcheilognathus lanceolata, A. limbata, A. signifer andTanakia tanago. However, the shape of the ripe eggs ofP. himantegus differed from those of the four species. As regards the shape of eggs, there was a common characteristic amongP. himantegus, Rhodeus uyekii andA. limbata from Korea. As regards larval development,P. himantegus had two characters also found inRhodeus. These facts seem to suggest thatP. himantegus is closely related toA. lanceolata, A. limbata, A. signifer andT. tanago but is more specialized than these four species, except forA. limbata from Korea.     

Crystallization of a complex between ribonuclease Ms and 3'-guanylic acid

The crystals of a complex between ribonuclease Ms, the extracellular ribonuclease from Aspergillus saitoi, and 3'-guanylic acid were obtained from 2-methyl-2,4-pentanediol solution by vapor diffusion technique in the hanging drop mode. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with dimensions a = 47.0 A, b = 62.8 A, c = 37.9 A. The crystals diffract strongly up to at least 2.0 A resolution.     

Inhibition of Endothelial Cell Differentiation on a Glycosylated Reconstituted Basement Membrane Complex

The vascular basement membrane is involved in the regulation of endothelial cell differentiation. The accumulation of advanced glycosylation endproducts (AGEs) has been demonstrated on these basement membranes in patients with diabetes. We examined the effect of AGEs on endothelial cell behavior on reconstituted basement membrane, Matrigel. Human umbilical vein-derived endothelial cells (HUVECs) stopped proliferating and differentiated into capillary-like tube-shaped structures on Matrigel. Laminin antibody partially blocked this process. HUVECs cultured on glycosylated Matrigel, however, proliferated and formed a monolayer without tube formation. The inclusion of aminoguanidine, an inhibitor of AGE formation, during the glycosylation of Matrigel restored HUVEC differentiation. Although the laminin adsorbed onto the plastic culture wells promoted HUVEC attachment and spreading, glycosylated laminin reduced HUVEC attachment by 50% and abolished cellular spreading. These effects were restored by aminoguanidine. HUVEC attachment to glycosylated laminin was further reduced by AGE-modified albumin, poly I, acetylated low-density lipoprotein, or maleylated albumin, ligands for a scavenger receptor. Coating the culture dishes with the laminin peptides RGD, YIGSR, and SIKVAV supported the attachment of HUVECs that was unaffected by glycosylation. Results suggest that AGE accumulation on the basement membranes inhibits endothelial cell differentiation by impairing the normal interactions of endothelial cell receptors with their specific matrix ligands. This process may be involved in diabetic angiopathy.     

A temperature-sensitive osmophilic mutant of Zygosaccharomyces rouxii

Summary A temperature-sensitive osmophilic mutant of Zygosaccharomyces rouxii, OS15, was isolated, which required high salt or sugar concentration for growth above 30°C. Cell viability at 35°C in the presence of NaCl was higher than in the absence of NaCl, and a survival ratio of the mutant cells after incubation at 55°C was also higher in the presence of NaCl than NaCl-free condition. Furthermore, resistance to UV light, hygromycin B and geneticin was improved in the presence of NaCl. There was no difference between the parent and the mutant in fatty acid saturation and microscopic cell shape under NaCl condition.     

Effects of endothelin-1 and conversion of big endothelin-1 in the isolated perfused rabbit lung.

We examined the effects of endothelin-1 (ET-1) on pulmonary hemodynamic and transvascular fluid filtration and the conversion of big endothelin-1 (big ET-1), a precursor of ET-1, in isolated perfused rabbit lungs at constant vascular and airway pressures. Furthermore we examined whether ET-1 contributes to cyclooxygenase metabolism. The perfusate flow decreased significantly after bolus administration of 1 or 0.1 nmol of ET-1. Lung weight did not increase throughout the experimental period. Big ET-1- (1 nmol) induced decrease in the flow was slow in developing, although the maximum response was comparable to that induced by the same dose of ET-1. The concentration of bit ET-1 in the perfusate progressively decreased, while that of ET-1 increased in a time-dependent manner. Phosphoramidon, an inhibitor of metalloproteinase, suppressed the pressor effect of big ET-1 (P less than 0.01) and the increase in the concentration of ET-1 in the perfusate (P less than 0.05). The present findings provide the first evidence suggesting that the potent vasocontractile effect of big ET-1 in pulmonary circulation can be attributed to the production of ET-1 by the conversion from big ET-1 in the vascular bed. ET-1-induced perfusate flow changes were not affected by indomethacin, and the concentration of 6-ketoprostaglandin F1 alpha, a metabolite of prostacyclin, did not increase after ET-1 administration.     

Crystal structure of the complex of subtilisin BPN' with its protein inhibitor Streptomyces subtilisin inhibitor. The structure at 4.3 Angstroms resolution

The crystal structure of the complex of subtilisin BPN′ (EC 3.4.21.14) with its protein inhibitor (Streptomyces subtilisin inhibitor) was solved at 4.3 Å resolution, thus establishing the following. (1) Two subtilisin BPN′ molecules (2E) associate with one dimeric inhibitor molecule (I₂) to form the complex molecule E₂I₂. (2) The conformation of neither the inhibitor nor subtilisin BPN′ undergoes any detectable change at this resolution upon complex formation. (3) The inhibitor binds to subtilisin to form an antiparallel β-sheet, as in the case of trypsin/ trypsin inhibitor complexes. (4) The scissible bond of the inhibitor is between Met73′ and Val74′, as proposed earlier (Ikenaka et al., 1974). (5) The protein inhibitor and the substrates bind to subtilisin BPN′ in essentially the same way.     

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.     

Multiple Neurite Formation in Neuroblastoma Cell Lines by Griseolic Acid, a Potent Inhibitor of Cyclic Nucleotide Phosphodiesterases

The morphological change of several neuroblastoma cell lines induced by griseolic acid, a novel and potent inhibitor of cyclic nucleotide phosphodiesterase (PDE), was examined. In the cell lines tested, Neuro-2a (a murine neuroblastoma cell line) showed dose-dependent (1 microM-1 mM) neurite extension. Griseolic acid markedly increased the intracellular cyclic AMP level of Neuro-2a cells, suppressed DNA synthesis (82% at 1 mM), and induced multipolar (multiple-neurite-bearing)-type neuritogenesis. A similar type of neurite outgrowth was induced by 8-bromo-cyclic AMP, which also elevated the intracellular cyclic AMP concentration. In contrast, when Neuro-2a cells were treated with retinoic acid, neurite formation was of the monopolar (single-neurite-bearing) type. Papaverine and theophylline, which have been frequently used as PDE inhibitors, failed to induce these morphological changes up to 1 mM, probably owing to the lesser potency of these compounds as compared with griseolic acid on the inhibition of PDE. Retinoic acid, theophylline, and papaverine were ineffective at elevating the intracellular cyclic AMP level. These results suggest that multipolar-type cell shape change in Neuro-2a cells is correlated with the accumulation of intracellular cyclic AMP and that griseolic acid is a useful compound to induce neuroblastoma cells into terminal differentiation.     

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.