Affinity labelling of endothelin receptor and characterization of solubilized endothelin-endothelin-receptor complex

Chick cardiac membranes were affinity labelled by cross-linking to membrane-bound 125I-endothelin-1 with disuccinimidyl tartarate. SDS/PAGE and autoradiographic analysis of the 125I-endothelin-1-labelled material in the presence or absence of 2-mercaptoethanol revealed one major labelled band, corresponding to a molecular mass of 53 kDa, whose appearance was dose-dependently inhibited by the addition of unlabelled endothelin-1 (1-100 nM). Subtracting the molecular mass of 125I-endothelin-1 and disuccinimidyl tartarate, the binding protein appeared to have a molecular mass of 50 kDa. To investigate further the molecular properties of endothelin receptor, the 125I-endothelin-1-endothelin-receptor complex was solubilized from chick cardiac membranes using the detergent digitonin. Sucrose gradient sedimentation of the solubilized complex indicated a sedimentation coefficient of 13 S, whereas the complex of (+)-[3H]PN200-110, a dihydropyridine derivative, and dihydropyridine-sensitive Ca2+ channels sedimented at 22 S. A monoclonal antibody raised against dihydropyridine-sensitive Ca2+ channels from the chick brain did not immunoprecipitate the 125I-endothelin-1-endothelin-receptor complex. These data suggest that endothelin receptor is clearly distinct from dihydropyridine-sensitive Ca2+ channels and endothelin has its own specific 50-kDa receptor.     

Isolation of anti-endothelin receptor monoclonal antibodies for use in receptor characterization

Monoclonal antibodies reactive with endothelin (ET) receptors have been prepared by immunization of mice with rat lung membranes. Of four clones isolated, three clones preferentially recognized 32,000-dalton ET receptor and the other has a higher affinity for the 45,000-dalton receptor. The binding of 125I-ET-1 to detergent-solubilized ET receptors which were adsorbed to the antibodies was displaced by increasing concentrations of unlabeled ET isopeptides. These results demonstrate that the four clones specific for the receptor have the potential to be a useful tool in the characterization of ET receptors.     

Cell-division inhibitor produced by a killer strain of cellular slime mold Polysphondylium pallidum

The killer strain CK-8 of cellular slime mold Polysphondylium pallidum produces a cell-division inhibitor, in addition to a killer factor. This inhibitor represses cell division of many strains and species of cellular slime molds, except CK-8 itself and its complementary mating-type strain. It is sensitive to both heat and trypsin, and capable of binding to Con A. Its apparent molecular mass is more than 100 kDa. Repression of cell division by this inhibitor is reversed by trypsin treatment of the cells.     

A possible new mechanism of temperature dependence of electron transfer in photosynthetic systems

A new theory for the electron transfer by the non-adiabatic process is formulated taking into account the origin shift and the frequency change of the vibration. The resultant formulas are quite similar to those of Jortner (Jortner, J. (1976) J. Chem. Phys. 64, 4860–4867) except that the free energy gap ΔG is used instead of the energy gap ΔE. By applying this theory to the photosynthetic electron transfer, the role of the remarkable temperature dependence of the electron transfer from cytochrome to P⁺ in Chromatium vinosum and the experimental data were reproduced very well using a small value of the coupling strength in contrast with the previous theory. This implies that proteins play a role to exclude many of the solvent molecules from the region of the electron transfer reaction between the donor and acceptor molecules. The negative activation process in the back electron transfer from Q^?_A to P⁺, the very slow back electron transfer from I^? to P⁺ and the solvent isotope effect on the cytochrome oxidation are also successfully explained by this new theory. It is shown that even a qualitative conclusion as to the molecular parameters obtained from the temperature dependence of the electron transfer is different between the present theory and that of Jortner.     

Corn Agmatine Iminohydrolase: PURIFICATION AND PROPERTIES

Agmatine iminohydrolase (EC 3.5.3.12) was purified 7,300-fold from extracts of corn shoots by chromatographic separations on diethylaminoethyl-cellulose, Sephadex G-100, and agmatine-affinity column. The enzyme was homogeneous by the criteria of analytical gel electrophoresis. Molecular weight estimated by Bio-Gel P-200 was 85,000, and the enzyme seems to be a dimer with identical subunits (molecular weight, 43,000). The isoelectric point determined by gel electrofocusing was 4.7. The optimal pH and temperature for activity were 6.5 and 60 C, respectively. The activation energy was 10.9 kilocalories per mole. High specificity exists for agmatine, the K_m value for agmatine was 1.9 × 10⁻⁴ molar, and the enzyme was present in the cytosol. The enzyme was sensitive to Cu²⁺ and Zn²⁺ and also was inhibited by p-hydroxymercuribenzoate and arcain.     

Structural abnormalities of the Y chromosome and abnormal external genitals

Summary Three infants with different types of Y-chromosome abnomalies, including short- and/or long-arm deletion and mosaicism, are reported. The karyotypes of these patients were: 45,X/46,X,del(Y)/47,X,del(Y), del(Y) on peripheral lymphocytes and 45,X/46,X, del(Y) on gonadal tissue (case 1), 45,X/46,X,del(Y) (case 2), and 45,X/46,X,r(Y) (case 3). In case 1 the euchromatic segment on the deleted Y was distinctly larger than that of the father's Y.The three infants had no gross phenotypic anomalies except ambiguous genitals and low birth weight, and they were small for date. The histologic diagnosis in two of them was mixed gonodal dysgenesis (cases 1 and 2).The relationship between structural abnormalities of the Y chromosome and ambiguous genitals as well as male-determining factors is discussed.     

Establishment of Autoantibody-Producing Cell Lines from Peripheral Blood Lymphocytes of Patients with Systemic Lupus Erythematosus

Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.     

Contribution of microsomal cytochrome b5 electron transport system coupled with Δ5-3β-hydroxysteroid dehydrogenase to androgen synthesis from progesterone

Cytochromes P-450 and $\text{b}$₅ were observed in the microsomal fraction of interstitial tissue of rat testes. Microsomal cytochrome $\text{b}$₅ was reduced by the NADH coupled with the activities of Δ⁵-3β-hydroxysteroid dehydrogenase with Δ⁵-Δ⁴ isomerase through conversion of pregnenolone to progesterone. Activities of NADPH-supported 17α-hydroxylase and C-17-C-20 lyase which converted progesterone to androstenedione were stimulated by either the presence of NADH or the oxidative reaction by the dehydrogenase upon Δ⁵-3β-hydroxysteroids. Androstenedione production enhanced by the reaction of the dehydrogenase was decreased by addition of the antibody against NADH-cytochrome $\text{b}$₅ reductase which was purified from rat hepatic microsomes, suggesting the active participation of cytochrome $\text{b}$₅ in the androgen synthesis.     

Effect of glucose on protein phosphorylation in rat pancreatic islets

Isolated rat pancreatic islets, incubated in the presence of extracellular ³²Pi to steady state ³²P incorporation into cellular phosphopeptides, were exposed to glucose for 10 min. Glucose (16.7 mM) significantly stimulated the phosphorylation of six phosphoproteins with molecular weights of 15,000, 35,000, 49,000, 64,000, 93,000 and 138,000. Mannoheptulose (16.7 mM) markedly inhibited glucose-stimulated phosphorylation of these six phosphoproteins. This protein phosphorylation might be important in mediating glucose-stimulated insulin release.     

Effects of the Brachyury (T) mutation on morphogenetic movement in the mouse embryo

$\text{T}\text{T}$ embryos have been first distinguishable at 8 days post coitum by their gross morphological abnormalities. By quantitative morphometry of histological sections, anomalies in the homozygotes were expressed numerically. At 8 days p.c., morphologically identifiable T-homozygotes had an increased number of ectodermal and a reduced number of mesodermal cells compared to the wild type. At 7 days p.c., embryos with a low mesoderm/ectoderm ratio were found only in litters of $\text{T}\text{+}\text{×}\text{T}\text{+}$ matings at the expected frequency. At 6 days p.c., one-fourth of the embryos in $\text{T}\text{+}\text{×}\text{T}\text{+}$ litters showed a delay in the transition from cuboidal to squamous endoderm. No such embryos were found in the +/+ × +/+ matings. In 6-, 7-, and 8-day mutant embryos, cells proliferated at statistically normal rates. Therefore, it may be said that advanced morphological irregularities of 8-day homozygotes cannot be accounted for by anomalies in cell proliferation. When the total cell number was 5 × 10⁴/embryo (8 days), a sudden change was observed in the regional distribution of mesodermal and ectodermal cells along the anteroposterior axis of $\text{T}\text{T}$ embryos. Since no regional difference in the cell cycle time was observed, these abnormalities may best be explained by anomalies in cell migration. These results strongly suggest abnormal morphology of $\text{T}\text{T}$ mutants resulting from defects in morphogenetic movement.