Specific reduction of delta-opioid receptor binding in transfected NG108-15 cells.

We have recently identified and sequenced the cDNA for an opioid-binding protein with homologies to cell adhesion molecules (OBCAM) (Schofield, P. R., McFarlard, K. C., Hayflick, J. S., Wilcox, J. N., Cho, T. M., Roy, S., Lee, N. M., Loh, H. H., and Seeburg, P. H. (1989) EMBO J. 8, 489-495). Several lines of evidence using antibodies suggest that OBCAM may play a functional role in NG108-15 neuroblastoma x glioma cells, a useful model system that contains a homogeneous population of delta-opioid receptors. A logical extension of this research is to further test this hypothesis. As part of this study, NG108-15 cells were stably transfected with either sense or antisense sequences of a portion of pROM, the rat cDNA for OBCAM. [3H] Diprenorphine binding was greatly reduced in antisense-transfected cells relative to non-transfected cells. Binding to alpha 2-adrenergic, muscarinic, and insulin receptors was unaffected. These results further support the notion that OBCAM or its analogue is part (or a subunit) of an opioid receptor. Furthermore, our observation of an apparently specific reduction in opioid binding in these transfected cells suggests that they may provide a novel genetic approach for studying regulation of the opioid receptor in this defined cell line.     

Induction of an organ-specific autoimmune disease, lymphocytic hypophysitis, in hamsters by recombinant rubella virus glycoprotein and prevention of disease by neonatal thymectomy.

Glycosylated, membrane-associated E1 (58-kDa) and E2 (47- to 49-kDa) rubella virus proteins and unglycosylated nucleoprotein C (33 kDa), from separately expressed vaccinia virus recombinants, were injected into golden Syrian hamsters. Rubella virus E1 and E2 glycoproteins consistently induced an organ-specific autoimmune disease, autoimmune lymphocytic hypophysitis, which was evidenced by the induction of autoantibodies against pituitary cells and by lymphocytic infiltration of the pituitary. Neonatal thymectomy prevented the disease. In contrast, rubella virus nucleoprotein C did not induce either autoantibodies against pituitary cells or lymphocytic infiltration of the pituitary. This finding raises the possibility that virus-specific protein itself can induce an organ-specific autoimmune disease in certain circumstances.     

A comparison of the frequency and type of chromosomal abnormalities in human sperm after different sperm capacitation conditions.

Human sperm karyotypes can be prepared after fusion of human sperm with Golden hamster oocytes. Most laboratories use one of two methods of sperm capacitation: incubation of freshly-ejaculated sperm in Biggers, Whitten, and Whittingham (BWW) medium for 5-7 h at 37 degrees C or sperm storage in (N-tris [hydroxymethyl]methyl-2-aminoethanesulfonic acid; 2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid) (TES)-Tris yolk buffer (TYB) for 1-3 days at 4 degrees C. Since there have been conflicting reports as to whether there is a difference in the frequency of structural chromosomal abnormalities between BWW capacitation and storage in TYB for 2 days, we analyzed a larger number of karyotypes (8974) from 136 donors to determine if there was any difference in the frequency or type of chromosomal abnormalities in sperm treated by fresh BWW capacitation, storage in TYB for 1 day (TYB-1), or storage in TYB for 2 days (TYB-2). There was no difference in the frequency of numerical chromosomal abnormalities or sex ratio in any of the three treatment groups. However, there was a significantly increased frequency of structural chromosomal abnormalities after storage in TYB-1 and TYB-2. There was no difference in the frequency or type of structural chromosomal abnormalities after sperm storage in TYB-1 compared to TYB-2.     

The carbohydrases of Bacillus stearothermophilus NCIB 11412 and NCIB 10814

Summary Two strains (NCIB 11412 and NCIB 10814) of the thermophilic organism Bacillus stearothermophilus were found to produce complex carbohydrase systems. The enzyme activities in each system include -amylase as the major component, maltase, pullulanase, a minor amylase and cyclodextrinase. The latter three activities are produced in low yield in both strains. A crude enzyme preparation from each strain possessed maltogenic properties on hydrolysis of soluble starch. Following rigorous purification procedures, the purified major -amylase from either strain did not produce maltose as a major end-product of starch hydrolysis. However, a partially purified mixture of pullulanase, minor amylase and cyclodextrinase activities from NCIB 11412 and NCIB 10814 produced 56.4% and 62.0% maltose, respectively, from soluble starch.     

Factors Affecting Germination of Oospores of Phytophthora infestans

When oospores from the pairing between A¹ and A² mating types of Phytophthora infestans were treated with 0.25 % KMnO₄ solution for 15 min and incubated at 19 °C under light on a modified S+L medium, germination commenced within 4 days and reached about 70 % after 20 days. Under these conditions, more than 25 % of oospores obtained from a 4-day-old culture germinated. To obtain a high germination rate of P. infestans oospores, light was essential during germination but not during growth and oospore formation. The optimum time for activation of oospores with 0.25 % KMnO₄ was 15 to 30 min and a suitable concentration of KMnO₄ for 15 min activation was 0.25 to 0.45 %.     

Kinetic characterization of a prestart cell division control step in yeast. Implications for the mechanism of alpha-factor-induced division arrest

In Saccharomyces cerevisiae yeast, a cycloheximide-sensitive "prestart" step (Ki = 0.12 +/- 0.05 microM) is passaged greater than or equal to 4 to less than or equal to 13 minutes prior to the alpha-factor-sensitive "Start" step in the cell cycle of normal proliferating cells and in the first and second bud emergence cycles of abnormally large cells that had been arrested for cell division with alpha-factor and allowed to recover. This identifies the chronologically last protein synthetic step of the cell cycle that occurs prior to the completion of Start. This step is named the last synthetic prestart or LSP step. Cells require the completion of the LSP step before they can perform Start during recovery from arrest by alpha-factor. Yet alpha-factor is known to prevent cell division by acting at Start. The combined data suggest that alpha-factor prevents the Start step of cell division by inactivating a protein that is 1) required for the performance of Start, and 2) synthesized shortly prior to Start in the last synthetic prestart step.     

Phenylalanine ammonia-lyase: Purification and characterization from soybean cell suspension cultures

Soybean cell suspension cultures (Glycine max L. cv. Kanrich) grown on high-nitrogen medium produce 50 mU/g fresh wt of phenylalanine ammonia-lyase [EC 4.1.3.5] 7–9 days after inoculation. Nitrate was not limiting when the peak of enzyme activity was reached. Phenylalanine ammonia-lyase was purified 53-fold to essentially electrophoretic homogeneity from cell extracts with 10% recovery. The enzyme was stable in crude extracts and through most stages of purification. No activity could be detected with tyrosine as substrate in either crude extracts or purified enzyme. The electrophoretic mobility was somewhat less than that of the enzyme from maize but both eluted from an agarose column at the same position and the molecular weight of the subunit was similar for both enzymes. Thus the soybean enzyme is composed of four subunits and the native enzyme is ~330,000 M_r. The variation in structure and/or size and availability of hydrophobic regions among phenylalanine ammonia-lyases from four sources (potato, maize, Rhodotorula glutinis, and soybean) was shown by the different elution patterns they exhibited on columns of ω-aminoalkyl agarose (agarose-C_n-NH₂, n = 0 to 8). The order of increasing hydrophobicity is soybean, potato, maize, R. glutinis. The soybean enzyme exhibited negative cooperativity before hydroxylapatite chromatography and positive cooperativity afterward. This is the first example of positive cooperativity observed for phenylalanine ammonia-lyase.     

XY female mice express H-Y antigen

Karyotypically XY individuals of the C57BL/6J-Y^POS mouse stock develop as females or hermaphrodites, but never as normal males. The aberrant sexual development results from the interaction of the C57BL/6J genetic background with the M. poschiavinus-derived Y chromosome. XY females from this stock were assayed for H-Y antigen. By the criteria of skin-grafting, the cell-mediated lympholysis test, and the popliteal lymph node assay, these XY females are antigenically indistinguishable from normal C57BL/6 males. Implications for the hypothesis that H-Y antigen induces formation of the mammalian testis are discussed.     

Chicken liver fatty acid synthetase dimer: Evidence of asymmetrical structure from iodoacetamide inhibition studies

The condensing component of chicken liver fatty acid synthetase is inhibited by a sulfhydryl reagent, iodoacetamide, with a second-order rate constant of 0.23 M^–1 sec^–1 at pH 7.0 and 0. Complete inactivation requires the modification of approximately 8-SH groups per dimer of the enzyme. Quantitation of the extent of inactivation in the presence of i mM acetyl CoA (which completely protects the enzyme against inactivation) and in its absence shows that complete inactivation results from the binding of approximately 1.1 tool of carboxamidomethyl residues per dimer. These data are consistent with the proposed functional asymmetry of the enzyme.