Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.     

Position and orientation in the metaphase equator of an interchange quadrivalent of hybrid rye

The position and orientation of an interchange quadrivalent in flattened lateral views of metaphase I were studied in pollen mother cells of hybrid rye. Five quadrivalent types showed three positional distributions in the equator, these distributions having elements both similar to and very different from those found previously for an interchange quadrivalent of Allium triquetrum.     

Four- and five-coordinate species in nickel-reconstituted hemoglobin and myoglobin: Raman identification of the nickel-histidine stretching mode

Nickel(II)-reconstituted hemoglobin (NiHb) and myoglobin (NiMb) and model Ni porphyrins have been investigated by Soret-resonance Raman difference spectroscopy. Two sets of frequencies for the oxidation-state and core-size marker lines in the region from 1300 to 1700 cm-1 indicate two distinct sites in NiHb. Only one of these sites is evident in the Raman spectra of NiMb. This result is consistent with the UV-visible absorption spectrum of NiHb, which shows two Soret bands at 397 and 420 nm and one Soret at 424 nm for NiMb. Excitation at the blue Soret component of NiHb with 406.7-nm laser radiation preferentially enhances the set of Raman marker lines typical of Ni-protoporphyrin IX [Ni(ProtoP )] in noncoordinating solvents. The wavelength of the blue Soret component and the Raman spectrum indicate four-coordination for this site in NiHb. Laser excitation in the red Soret band enhances a set of lines whose frequencies are compatible with neither four- nor six-coordinate frequencies but are intermediate between the two. The red Soret band of the proteins is also considerably less red shifted than six-coordinate Ni-porphyrin models. These results suggest that Ni in the second site possesses a single axial ligand. Raman spectra of 64Ni-reconstituted and natural abundance Ni-reconstituted hemoglobins, obtained simultaneously in a Raman difference spectrometer, have identified the Ni-ligand stretch at 236 cm-1. The line shifts to 229 cm-1 for the 64Ni-reconstituted Hb. For a pure Ni-ligand stretch a 10-cm-1 shift would be predicted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Target-sensitive immunoliposomes: preparation and characterization

A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a protein encoded by the trans activator gene tatIII of human T-cell lymphotropic retrovirus type III.

The human T-cell lymphotropic virus type III (HTLV-III/LAV) is a retrovirus associated with acquired immune deficiency syndrome. The region on the viral genome that is necessary for trans-activation of the HTLV-III/LAV long terminal repeat called tatIII has previously been determined to lie between nucleotides 5365 and 5607. Here we report that a bacterial fusion protein containing amino acid sequences specified by the first coding exon of the tatIII gene is recognized by some patient antisera. We also demonstrate that lymphoid and epithelial cells that express the trans activator function express a 14-kilodalton (kDa) protein recognized by a patient antiserum that reacts with the bacterial tatIII fusion protein. Cells transiently transfected with a deletion mutant of the trans activator protein produce a 12-kDa protein rather than the 14-kDa protein. These observations indicate that the tatIII region contains a functional gene and is capable of expressing a protein that migrates with an apparent molecular size of 14 kDa in some lymphoid and epithelial cells transfected with plasmids containing the tatIII region. We propose that the product of the trans activator gene be designated p14tat-III.     

Transformation of Aspergillus nidulans with a cloned, oligomycin-resistant ATP synthase subunit 9 gene

Summary An allele (oliC31) of the A. nidulans oliC gene has been cloned using homology with the equivalent gene from N. crassa. OliC31 codes for an oligomycin-resistant, triethyltin-hypersensitive form of subunit 9 of the mitochondrial ATP synthase complex. Direct selection for oligomycin-resistance was possible following transformation of A. nidulans with the oliC31 gene. The phenotypes of transformants cultured in the presence of oligomycin were indicative of the position of integration of the transforming plasmid within the genome. Subsequent recombination events involving the integrated oliC31 gene were also apparent from altered levels of resistance to oligomycin or triethyltin. This gene should prove useful as a marker for transformation of strains lacking auxotrophic lesions and in gene replacement or disruption experiments.     

Hormonal regulation of the development of protease and carboxypeptidase activities in barley aleurone layers

Carboxypeptidase and protease activities of hormone-treated barley (Hordeum vulgare cv Himalaya) aleurone layers were investigated using the substrates N-carbobenzoxy-Ala-Phe and hemoglobin. A differential effect of gibberellic acid (GA₃) on these activities was observed. The carboxypeptidase activity develops in the aleurone layers during imbibition without the addition of hormone, while the release of this enzyme to the incubation medium is enhanced by GA₃. In contrast, GA₃ is required for both the production of protease activity in the aleurone layer and its secretion. The time course for development of protease activity in response to GA₃ is similar to that observed for α-amylase. Treating aleurone layers with both GA₃ and abscisic acid prevents all the GA₃ effects described above. Carboxypeptidase activity is maximal between pH 5 and 6, and is inhibited by diisopropylfluorophosphate and p-hydroxymercuribenzoate. We have observed three protease activities against hemoglobin which differ in charge but are all 37 kilodaltons in size on sodium dodecyl sulfate polyacrylamide gels. The activity of the proteases can be inhibited by sulfhydryl protease inhibitors, such as bromate and leupeptin, yet is enhanced by 2-fold with 2-mercaptoethanol. In addition, these enzymes appear to be active against the wheat and barley storage proteins, gliadin and hordein, respectively. On the basis of these characteristics and the time course of GA₃ response, it is concluded that the proteases represent the GA₃-induced, de novo synthesized proteases that are mainly responsible for the degradation of endosperm storage proteins.     

Barley aleurone layers secrete a nuclease in response to gibberellic Acid : purification and partial characterization of the associated ribonuclease, deoxyribonuclease, and 3'-nucleotidase activities

Incubation of barley (Hordeum vulgare L. cv Himalaya) half-seeds with gibberellic acid enhances the secretion of ribonuclease and deoxyribonuclease from aleurone tissue (MJ Chrispeels, JE Varner 1967 Plant Physiol 42: 398-406; L Taiz, JE Starks 1977 Plant Physiol 60: 182-189). These activities were over 50-fold greater in medium of half-seeds incubated with gibberellic acid than in control medium. Ribonuclease and deoxyribonuclease activities initially appeared in the medium 24 to 48 hours after hormone induction and increased for up to 96 hours. Both activities had a pH optimum of 6.0 and a temperature optimum of 55°C. When the medium from gibberellic acid-treated half-seeds was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the major ribonuclease and deoxyribonuclease activity bands comigrated. The two enzyme activities remained associated throughout a 2,700-fold purification employing ammonium sulfate fractionation, Heparin-Agarose affinity chromatography, and Reactive Blue 2-Agarose affinity chromatography. Also accompanying the ribonuclease and deoxyribonuclease activities throughout purification was the ability to hydrolyze the 3′-phosphoester linkage of 3′-AMP. The purified protein was composed of a single polypeptide with an apparent molecular weight of 36 kilodaltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is concluded that in response to gibberellic acid, barley aleurone tissue secretes a nuclease having ribonuclease, deoxyribonuclease, and 3′-nucleotidase activities.     

Characterization of a DNA repair domain containing the dihydrofolate reductase gene in Chinese hamster ovary cells

The formation and removal of UV-induced pyrimidine dimers were measured in restriction fragments near and within the essential dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells in order to map the genomic fine structure of DNA repair. Dimer frequencies were determined at 0, 8, and 24 h after irradiating the cells with 20 J/m2 UV light (254 nm). Within 8 h, the cells had removed more than 40% of the dimers from sequences near the 5' end of the gene, somewhat fewer from the 3' end, but only 2% from the 3' flanking region and 10% from a region upstream from the gene. The corresponding extent of repair in the genome as a whole is 5-10% in the 8-h period. Isoschizomeric restriction enzyme analysis was used to detect the level of methylation in the fragments in which repair was measured. We found that the only hypomethylated sites in and around the DHFR gene were in the fragment near its 5' end, which displayed maximal DNA repair efficiency. The size of the region of preferential DNA repair at the DHFR locus appears to be in the range of 50-80 kilobases, and this finding is discussed in relation to genomic domains and the structure of mammalian chromatin.