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.     

Elevation of Cyclic AMP-Dependent Protein Kinase Activity during Migration of Primary Mesenchyme Cell in Sand Dollar Blastulae

Concetration of intracellular cyclic AMP (cAMP), and activities of adenylate cyclase and cAMP-dependent protein kinase were examined in swimming and mesenchyme blastulae and primary mesenchyme cells (PMCs) of the sand dollar, Clypeaster japonicus , respectively. In mesenchyme blastulae, the concentration of cAMP increased 45% from that in swimming blastulae. PMCs contained a concentration of cAMP 40% higher than that in whole embryos at the mesenchyme blastula stage. The activity of adenylate cyclase in mesenchyme blastulae was 100% higher than that in swimming blastulae. The activites of cAMP-dependent protein kinase in whole embryos at the above two developmental stages, on the other hand, were quite similar to each other. However, in PMCs the activity of the enzyme was conspicuously higher than that in these embryos, and it reached 190% higher than that in these embryos. Inhibition of cAMP-dependent protein kinase activity by a synthetic inhibitor, H8, caused severe inhibition of PMC migration but it did not exert any effect on PMC ingression. These results suggest that the cAMP-dependent protein kinase activity is involved in PMC migration, but not in PMC ingression.     

Calcium binding to phospholipid: structural study of calcium glycerophosphate.

To consider possible interaction of the phospholipid membrane with calcium ions, crystal structures of calcium dl-alpha- and beta-glycerophosphates (alpha- and beta-CaGs, respectively) were investigated by X-ray diffraction methods. After many attempts, relatively large single crystals of beta-CaG were prepared from the aqueous solution containing HCl, while crystals of CaHPO4.2H2O were obtained from alpha-CaG solution under the same crystallization conditions. The crystal structure of beta-CaG is orthorhombic with space group Pna2(1) and cell dimensions of a = 8.251(1), b = 13.038(3), c = 25.483 (10) A, V = 2741.5 (13) A3 and Z = 16 [four molecules (A to D) in an asymmetric unit]. Molecules of A to D took, as a whole, similar extended conformations, although A and B were different from C and D in the orientation about a glycerol C-C bond. Four independent beta-glycerophosphates commonly act as two types of bidentate ligands, where one is the coordination to the calcium ion by the glycerol O(1) and phosphate O(22) atoms, and the other by the phosphate O(22) and O(23) atoms, thus forming the calcium coordination of a distorted square plane, respectively. Each of four independent calcium ions forms the same coordination geometry of a distorted pentagonal bipyramid. Infinite double layers consisting of alternate A/B molecules and of alternative C/D ones and sandwiching calcium ions were arranged face-to-face along the b-direction and were piled up in the a-direction, thus forming the stacked bilayer unit with the thickness of d002 = 12.75 A. The elaborate networks of calcium coordinations and hydrogen bondings were formed among the layers and stabilized the crystal structure. Based on the structural parameters of the present beta-CaG crystal, a possible interaction model of phospholipid with calcium ions was proposed.     

X-ray crystallographic conformational study of 5'-O-[N-(L-alanyl)-sulfamoyl]adenosine, a substrate analogue for alanyl-tRNA synthetase

In order to elucidate the substrate specificity of alanyl-tRNA synthetase, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine (Ala-SA), an analogue of alanyl-AMP, was chemically synthesized. Its binding ability is similar to that of the substrate based on the inhibitory activity for the aminoacylation of alanyl-tRNA synthetase. Taking advantage of the stable sulfamoyl bond of Ala-Sa, compared with the highly labile aminoacyl bond of alanyl-AMP, the molecular conformation of the former inhibitor was studied by X-ray single crystal analysis. Crystal data are as follows: C13H19N7O7S.2H2O, space group C2, a = 39.620(6), b = 5.757(1), c = 20.040(3) A, beta = 117.2(1) degrees, V = 4065(9) A3, Z = 8, and final R = 0.065 for 2785 independent reflections of F(2)0 greater than or equal to 2 sigma (F0)2. In the crystal, the molecule is in a zwitterionic state with the terminal amino group protonated and sulfamoyl group deprotonated, and takes an open conformation, where the L-alanine moiety is located far from the adenosine moiety with gauche/trans and trans orientations about the exocyclic C(4')-C(5') and C(5')-O(5') bonds, respectively. The conformation of the adenosine moiety is anti for the glycosyl bond and C(3')-endo for the ribose puckering, and alanine is in the usually observed trans region for the psi torsion angle. The molecular dimensions of the sulfamoyl group are nearly the same as those of the phosphate group. The biological significance of the observed Ala-SA conformation is discussed in relation with the molecular conformation of tyrosyl-AMP complexed with tyrosyl-tRNA synthetase.     

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

In vivo and in vitro correlation of trachealis muscle contraction in dogs.

Maximal trachealis muscle shortening in vivo was compared with that in vitro in seven anesthetized dogs. In addition, the effect of graded elastic loads on the muscle was evaluated in vitro. In vivo trachealis muscle shortening, as measured using sonomicrometry, revealed maximal active shortening to be 28.8 +/- 11.7% (SD) of initial length. Trachealis muscle preparations from the same animals were studied in vitro to evaluate isometric force generation, isotonic shortening, and the effect of applying linear elastic loads to the trachealis muscle during contraction from optimal length. Maximal isotonic shortening was 66.8 +/- 8.4% of optimal length in vitro. Increasing elastic loads decreased active shortening and velocity of shortening in vitro in a hyperbolic manner. The elastic load required to decrease in vitro shortening to the extent of the shortening observed in vivo was similar to the estimated load provided by the tracheal cartilage. We conclude that decreased active shortening in vivo is primarily due to the elastic afterload provided by cartilage.     

Message amplification phenotyping of an inherited delta-aminolevulinate dehydratase deficiency in a family with acute hepatic porphyria

The molecular basis of the enzymatic defect responsible for acute hepatic porphyria due to delta-aminolevulinate dehydratase (ALAD) deficiency was investigated in a family including a proband with the acute disease. In order to delineate the mutation in the proband, cDNA for deficient ALAD was synthesized from the proband's cells. The ALAD phenotype was studied by message amplification phenotyping with total RNA extracted from lymphoblastoid cells of the proband and his family members. Two independent mutant alleles of ALAD were identified in the proband's cells. One mutant allele was shown to result in an amino acid substitution at residue 274 (Ala274----Thr). Message amplification phenotyping studies have also permitted us to define the ALAD phenotype of each subject in the family. This is the first mutation to be recognized in the human ALAD gene.     

The nucleotide sequence of the mouse immunoglobulin epsilon gene: comparison with the human epsilon gene sequence

We have determined the nucleotide sequence of the immunoglobulin epsilon gene cloned from newborn mouse DNA. The epsilon gene sequence allows prediction of the amino acid sequence of the constant region of the epsilon chain and comparison of it with sequences of the human epsilon and other mouse immunoglobulin genes. The epsilon gene was shown to be under the weakest selection pressure at the protein level among the immunoglobulin genes although the divergence at the synonymous position is similar. Our results suggest that the epsilon gene may be dispensable, which is in accord with the fact that IgE has only obscure roles in the immune defense system but has an undesirable role as a mediator of hypersensitivity. The sequence data suggest that the human and murine epsilon genes were derived from different ancestors duplicated a long time ago. The amino acid sequence of the epsilon chain is more homologous to those of the gamma chains than the other mouse heavy chains. Two membrane exons, separated by an 80-base intron, were identified 1.7 kb 3' to the CH4 domain of the epsilon gene and shown to conserve a hydrophobic portion similar to those of other heavy chain genes. RNA blot hybridization showed that the epsilon membrane exons are transcribed into two species of mRNA in an IgE hybridoma.     

The periodic change in adenosine 3′,5′-monophosphate concentration in sea-urchin eggs

The level of adenosine 3′,5′-monophosphate (cyclic AMP) in the eggs of the sea urchin, Anthocidaris crassispina, was found to change periodically after fertilization. The minimum and maximum levels of cyclic AMP were 1.0·10^?7 M and 1.5·10^?6 M, respectively. The activity of adenylate cyclase in a 105 000 × g precipitate reached a plateau at 20 min after fertilization and stayed constant for at least 2 h. It was also found that 1.0 mM CaCl₂ increased the activity of adenylate cyclase in the same precipitate from unfertilized eggs. In contrast, phosphodiesterase activity changed periodically and correlated with cyclic AMP levels in the eggs. Up to a concentration of 1.5·10^?6 M cyclic AMP, phosphodiesterase activity was low, but it became activated when the level of cyclic AMP rose beyond this level. These results indicate that the change in the intracellular level of cyclic AMP is regulated mainly by the change in phosphodiesterase activity.     

Potentiation by BL191 of differentiation of neuroblastoma cells induced by dibutyryl cAMP and prostaglandin E1

BL191, a newly developed phosphodiesterase inhibitor, markedly potentiated a differentiation of neuroblastoma cell clones (Neuro2a, NS-20Y, and N1E115) induced by dibutyryl cyclic adensoine 3′:5′-monophosphate(dibutyryl cAMP) and prostaglandin E₁ (PGE₁). BL191 (1 mM) inhibited DNA synthesis more strongly when used together with PGE₁ (0.5 μg/ml) and dibutyryl cAMP (0.5 mM) than papaverine (1.6 μg/ml) alone did. The inhibition rates of DNA synthesis were 72.5% for N1E-115, 75.3% for Neuro2a, and 82.5% for NS-20Y. After the treatment with BL191. PGE₁, and dibutyryl cAMP for 48 h all of three cell lines became enlarged and flattened, and extended long processes. The specific activities of choline acetyl transferase (EC 2.3.1.9) of NS-20Y and dopamine β-hydroxylase (EC 1.14.17.1) of N1E-115 increased about 3-fold as compared to the controls. The tumorigenicities of Neuro2a and N1E-115 cells were decreased, but not of NS-20Y. These data suggest the heterogenous responsiveness in neuroblastoma cells to drug treatment.