Polyadenylic acid synthesis activity of purified DNA-dependent RNA polymerase from Caulobacter

Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor.     

Failure of atractyloside to inhibit synaptosomal mitochondrial energy transduction

Studies on synaptosome mitochondrial respiration are complicated by “free” mitochondria. Veratridine stimulation of synaptosomal respiration was due to increased Na⁺ cycling at the synaptosome membrane associated with increased oxidative phosphorylation of intraterminal ADP and was inhibited by oligomycin, ouabain or Na⁺ free medium. Atractylate or carboxyatractyloside failed to block veratridine-stimulated respiration but inhibited exogenous-ADP-stimulated respiration. Protein synthesis in the synaptosome fraction was inhibited by oligomycin, valinomycin or 2,4-dinitrophenol but was unaffected by excess atractylate. No change in synaptosomal adenine nucleotide content was found in the presence of atractylate, although a significant decrease in the [ATP]/[ADP] was found with oligomycin, veratridine or valinomycin. These findings show that atractylate does not modify intraterminal mitochondrial energy transduction and indirectly suggest an impermeability of the synaptosome membrane to atractylate.     

Possible molecular detent in the DNA structure at regulatory sequences

A common feature that appears in a number of DNA sites where proteins interact is the sequence GTG/CAC. In the lac operator this sequence leads to a region with a higher imino proton exchange rate well below the optical melting temperature. It is suggested that this reflects a structural feature recognized by proteins that bind specific sites on the DNA molecule.     

Dynein ATPase is inhibited selectively in vitro by erythro-9-[3-2-(hydroxynonyl)]adenine

Dynein (ATP phosphohydrolase, EC 3.6.1.3) extracted from sea urchin sperm tails was inhibited by erythro-9-[3-2-(hydrosynonyl)]adenine in a dosedependent fashion; at the 50% inhibitory concentration, 0.23 mM, twelve other ATP-metabolizing enzymes were notsignificantly affected. Actomyosin and myosin ATPase activities were enhanced 1.5- to 2-fold by millimolar concentrations of erythro-9-[3-2-(hydroxynonyl)]adenine. Enzyme kinetic analysis supported a model of linear mixed-type inhibition, which suggests that the binding site for erythro-9-[3-2-(hydroxynonyl)]adenine on dynein is remote from the ATPase active site. As a selective inhibitor $\text{in}\text{vitro}$, erythro-9-[3-2-(hydroxynonyl)]adenine appears to offer a biochemical criterion for identifying dynein isozymes in tissue extracts.     

Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages

Studies with populations of macrophages have produced conflicting results concerning the possibility that the concentration of intracellular ionized calcium [( Ca2+]i) may act as an important mediator for phagocytosis. Since asynchronous changes in [Ca2+]i in individual cells undergoing phagocytosis may be averaged to undetectability in population studies, we studied single adhering murine macrophages using fura-2 and our previously described digital imaging system. The proportion of macrophages phagocytosing IgG-coated latex beads was greater than for uncoated beads (percent phagocytosing cells: 71 +/- 7 vs. 27 +/- 7, P less than 0.01). Phagocytosis of IgG-coated and uncoated beads was always associated with a calcium transient that preceded the initiation of phagocytosis. No calcium transients were detected in cells that bound but did not phagocytose beads. Four major differences between Fc receptor-mediated and nonspecific phagocytosis were detected: (a) the duration of calcium transients was longer for nonspecific phagocytosis compared with Fc receptor-mediated phagocytosis (69.9 +/- 10.2 vs. 48.7 +/- 4.7 s, P less than 0.05) and the magnitude of calcium transients was less for nonspecific phagocytosis (178 +/- 43 vs. 349 +/- 53 nM, P less than 0.05); (b) removal of extracellular calcium abolished the calcium transients associated with nonspecific phagocytosis but had no effect on those associated with receptor-mediated phagocytosis; (c) in the absence of extracellular calcium, buffering intracellular calcium with a chelator reduced Fc receptor-mediated phagocytosis but had no additive inhibitory effect on nonspecific phagocytosis; and (d) inhibition of protein kinase C (PKC) with staurosporine inhibited nonspecific phagocytosis but had no effect on receptor-mediated phagocytosis. Our observations suggest that despite both types of phagocytosis being associated with intracellular calcium transients, the role played by intracellular calcium in the signaling pathways may differ for Fc receptor-mediated and nonspecific phagocytosis by elicited murine macrophages.     

Chromatographic Evidence for the Presence of Multiple Tachykinins in the Bovine Adrenal Medulla

Among the mammalian tachykinins, substance P (SP) has been shown to be the most potent at modulating the response due to nicotinic acetylcholine receptor stimulation of bovine adrenal chromaffin cells. SP-like immunoreactivity has been detected in nerve terminals innervating the adrenal medulla; however, little is known of the presence of other tachykinins in this tissue. In this study, reverse-phase HPLC was used to fractionate peptides in bovine adrenal medullary extracts, and the fractions were analyzed by radioimmunoassay using antisera to SP or neurokinin A (NKA). The results show that both NKA- and SP-like immunoreactivities are present in the adrenal medulla. The presence of neurokinin B is also indicated. The presence of multiple tachykinins in this tissue raises questions as to their functions in the adrenal medulla.     

Interaction between high-density lipoprotein subpopulations in apo B-free and abetalipoproteinemic plasma.

Two populations of high-density lipoprotein (HDL) particles exist in human plasma. Both contain apolipoprotein (apo) A-I, but only one contains apo A-II: Lp(AI w AII) and Lp(AI w/o AII). To study the extent of interaction between these particles, apo B-free plasma prepared by the selective removal of apo B-containing lipoproteins (LpB) from the plasma of three normolipidemic (NL) subjects and whole plasma from two patients with abetalipoproteinemia (ABL) were incubated at 37 degrees C for 24 h. Apo B-free plasma samples were used to avoid lipid-exchange between HDL and LpB. Lp(AI w AII) and Lp(AI w/o AII) were isolated from each apo B-free plasma sample before and after incubation and their protein and lipid contents quantified. Before incubation, ABL plasma had reduced levels of Lp(AI w AII) and Lp(AI w/o AII), (40% and 70% of normals, respectively). Compared to the HDL of apo B-free NL plasma, ABL HDL had higher relative contents of free cholesterol, phospholipid and total lipid, and contained more particles with apparent hydrated Stokes diameter in the 9.2-17.0 nm region. These differences were particularly pronounced in particles without apo A-II. Despite their differences, the total cholesterol contents of Lp(AI w AII) increased, while that of Lp(AI w/o AII) decreased in all five plasma samples and the amount of apo A-I in Lp(AI w AII) increased by 6-8 mg/dl in four during the incubation. These compositional changes were accompanied by a relative reduction of particles in the 7.0-8.2 nm Stokes diameter size region and an increase of particles in the 9.2-11.2 nm region. These data are consistent with intravascular modulation between HDL particles with and without apo A-II. The observed increase in apo A-II-associated cholesterol and apo A-I, could involve either the transfer of cholesterol and apo A-I from particles without apo A-II to those with A-II, or the transfer of apo A-II from Lp(AI w AII) to Lp(AI w/o AII). The exact mechanism and direction of the transfer remain to be determined.     

Fiddlehead: an Arabidopsis mutant constitutively expressing an organ fusion program that involves interactions between epidermal cells.

In most circumstances plant epidermal cells do not respond to surface contact with adjacent plant parts. We have identified and characterized a mutant of Arabidopsis thaliana, designated fiddlehead, where lateral appendages of the shoot fuse with one another. While fusion between floral organs is most frequent, leaf fusions also occur. Using scanning and transmission electron microscopy, we show that adhesion takes place between epidermal cells and does not involve cytoplasmic union. We also show that the frequency of organ fusion is dictated by organ proximity. In wildtype Arabidopsis, postgenital fusion takes place exclusively in the gynoecium, whereas in the fiddlehead mutant, this program becomes expressed constitutively. The existence of such a mutant demonstrates that postgenital fusion is a genetically distinct program superimposed upon other aspects of gynoecial development in Arabidopsis.     

A specific and potent immunotoxin composed of antibody ZME-018 and the plant toxin gelonin

Murine monoclonal antibody ZME-018 recognizes a 240 Kda glycoprotein present on the surface of most human melanoma cells and on over 80% of human biopsy specimens tested. Gelonin is a ribosome-inactivating plant toxin similar in nature and rivaling the activity of ricin A chain. ZME-018 was coupled to purified gelonin using the reagents SPDP and 2-iminothiolane. The ZME-gelonin conjugate was purified by S-300 Sephacryl and Blue Sepharose chromatography, removing unreacted gelonin and antibody, respectively. PAGE analysis showed that ZME was coupled to 1, 2, or 3 gelonin molecules. The ZME-gelonin conjugate was 10(6)-fold more active than gelonin itself in inhibiting the growth of log-phase human melanoma cells in culture. The immunoconjugate was not cytotoxic to antigen negative T-24 (human bladder carcinoma) cells. Treatment of melanoma cells with recombinant IFN-alpha or TNF substantially augmented the cytotoxicity of the immunoconjugate while treatment with IFN-gamma had a minor effect. Using the human tumor colony assay of melanoma cells obtained from fresh biopsy specimens, greater than 90% growth suppression was observed in 2 of 4 samples tested at a concentration of 250 ng/ml. In addition, 25% growth suppression was observed with a third sample tested, and no growth suppression was observed in 1 sample. Thus, clonogenic melanoma cells are sensitive in vitro to the cytotoxic activity of this immunotoxin at concentrations which we presume are pharmacologically relevant.