Regulation of expression of glutamine synthetase in a symbiotic Nostoc strain associated with Anthoceros punctatus.

A characteristic of N2-fixing cyanobacteria in symbiotic associations appears to be release of N2-derived NH4+. The specific activity of the primary ammonium-assimilating enzyme, glutamine synthetase (GS), was found to be three- to fourfold lower in Nostoc sp. strain 7801 grown in symbiotic association with the bryophyte Anthoceros punctatus than in free-living Nostoc sp. strain 7801. Quantitative immunological assays with antisera against GS purified from Nostoc sp. strain 7801 and from Escherichia coli indicated that similar amounts of the GS protein were present in symbiotic (50 micrograms mg-1) and free-living (68 micrograms mg-1) cultures. The conclusion from these experiments is that GS is regulated by a posttranslational mechanism in Anthoceros-associated Nostoc sp. strain 7801. However, the results of comparative catalytic and immunological experiments between N2- and NH4+-grown free-living Nostoc sp. strain 7801 implied control of GS synthesis. A correlation was not observed between the level of GS expression and the extent of symbiotic heterocyst differentiation in Nostoc sp. strain 7801 associated with A. punctatus.     

Genetic counseling and prenatal diagnosis in Duchenne myopathy: yesterday, today and tomorrow

The coming of molecular biology has greatly modified the concept of genetic counselling and prenatal diagnosis of Duchenne muscular dystrophy. The most important stages of the genetic counselling are reported: estimate of the risk and carrier detection. This heterozygote detection is now possible in a few cases owing to polymorphic DNA markers recently identified that are genetically linked to the DMD gene locus and detected with probes. An analysis of foetal DNA is also possible and allows us to consider prenatal diagnosis of this affection. This study is yet limited by two impediments: on one hand low rate of informative families, on the other hand use of markers that are not very closely linked to DMD involving recombinations and risks of errors. The solution of these problems is in the use of linked DNA markers with the best polymorphism flanking the Duchenne muscular dystrophy locus. Finally the authors report the necessity of strict collaboration systems between clinical experts, geneticists, biologists and informaticians.     

Structural and dynamic properties of a fluorouracil-adenine base pair in DNA studied by proton NMR

An oligodeoxynucleotide duplex containing the chemotherapeutic agent 5-fluorouracil (FU) has been constructed by solid phase phosphotriester synthesis and has been studied in solution by proton NMR. In this study, we provide the first structural characterization of a DNA complex containing a FU.A base pair. It has been determined that the 7-mer duplex containing a central FU.A base pair adopts a normal right-handed configuration and the A residue in the FU.A pair is oriented in the normal anticonfiguration giving a Watson-Crick base pair. The significant difference between T.A and FU.A base pairs is dynamic, not structural: the FU.A base pair opens faster than normal base pairs in the oligonucleotide studied. We provide evidence that the FU.A base pair has a significantly enhanced opening rate resulting form decreased stacking of the 5-fluorouracil residue and not from the enhanced acidity of the 5'-fluorouracil imino proton.     

Purification and characterization of trichosanthin. Homology to the ricin A chain and implications as to mechanism of abortifacient activity

Trichosanthin, a protein from the Chinese medicinal herb Trichosanthes kirilowii, was purified in two essentially quantitative steps involving CM-Sephadex chromatography and reverse-phase high performance liquid chromatography. The protein was found to have a molecular mass of 25-26 kDa, to contain no cysteine, and to contain no glycosidic linkages. Pure trichosanthin was found to have potent abortifacient activity in pregnant mice. In order to understand the molecular basis of this unique biological activity, we have examined the amino acid sequence of the protein. As purified, trichosanthin was found to contain two amino-terminal sequences which differed only in the absence or presence of a tyrosine at residue 1. Sequence analysis of trichosanthin has allowed for determination of the NH2-terminal 38-amino acid residues. Comparison of this sequence to those present in a data base revealed homology with the ricin A-chain. Consistent with this structural homology, we have found that trichosanthin is a potent inhibitor of protein synthesis in a reticulocyte lysate system.     

Phorbol esters and calcium ionophores inhibit internalization and accelerate recycling of receptors in macrophages

Exposure of macrophages to phorbol esters or the calcium ionophore A23187 increases the number of several surface receptors due to recruitment of receptors from internal pools (Buys, S. S., Keogh, E. A., and Kaplan, J. (1984) Cell 38, 569-576). We have examined the mechanism by which these agents increase surface receptor number. Cells which were preloaded with either fluid phase or receptor-mediated ligands did not lose ligand following exposure to ionophore or phorbol ester. The rate of movement of ligands to the lysosome was also unaffected. These results suggest that A23187 does not induce the fusion of ligand-containing compartments with the cell surface. Ionophore treatment did, however, produce a severalfold increase in the rate at which unoccupied receptors reappear on the cell surface. These results suggest that the compartment of receptors affected by the ionophore formed subsequent to the dissociation of ligand from receptor. The altered rate of receptor reappearance was transitory (90 s), and the increase in receptor number was subsequently maintained by a decrease in the rate of internalization. Changes in the rate of receptor internalization did not correlate with changes in the rate of fluid phase pinocytosis, suggesting that the effect on receptor internalization was selective.     

Effect of phagocytosis on receptor distribution and endocytic activity in macrophages

Phagocytosis requires the internalization of a significant fraction of the plasma membrane and results in the intracellular deposition of large particles. We evaluated the effect of phagocytosis on the cellular distribution of recycling receptors and uptake of ligand to determine whether phagocytosis affects receptor behavior. Phagocytosis of zymosan, latex particles, or IgG-coated red blood cells by rabbit alveolar macrophages did not decrease the number of cell surface receptors for transferrin, alpha 2-macroglobulin X protease complexes, maleylated proteins, or mannosylated proteins. The number of surface receptors for transferrin was also unaltered in J774 cells, a macrophage-like cell line. In both cell types extensive phagocytosis did not affect the rate of receptor-mediated endocytosis or the distribution of receptors between the endosome and the cell surface. However, fluid phase pinocytosis was reduced by phagocytosis. The major reduction appeared to be not in the rate of internalization but rather in the delivery of fluid to the lysosome. These results demonstrate that internalization of a significant amount of the plasma membrane during phagocytosis does not diminish the number of receptors on the cell surface and has no effect on receptor-mediated ligand uptake.     

A Phorbol Ester-Sensitive Kinase Catalyzes the Phosphorylation of P0 Glycoprotein in Myelin

The proposed structural protein of peripheral nerve myelin, P0, has been shown to have several covalent modifications. In addition to being glycosylated, sulfated, and acylated, P0 is phosphorylated, with the intracellular site of this latter addition being in question. By employing nerve injury models that exhibit different levels of P0 biosynthesis in the absence and presence of myelin assembly, we have examined the cellular location of P0 phosphorylation. It is demonstrated that there is comparable P0 phosphorylation in both normal and crush-injured adult rat sciatic nerves, although the level of biosynthesis of P0 differs between these myelin maintaining and actively myelinating nerve models, respectively. The glycoprotein does not appear to be phosphorylated readily in the transected adult sciatic nerve, a preparation in which P0 biosynthesis is observed but that lacks myelin membrane. These observations suggest that the modification is not associated with the biosynthesis or maturation of P0 in the endoplasmic reticulum or Golgi, but that it instead occurs after myelin assembly. That P0 phosphorylation occurs in the normal nerve even when translation is inhibited by cycloheximide treatment lends further support to this conclusion. P0 is shown to be phosphorylated on one or more serine residues, with all or most of the phosphate group(s) being labile as evidenced by pulse-chase analysis. Addition of a biologically active phorbol ester, 12-O-tetradecanoylphorbol-13-acetate or 4 beta-phorbol 12,13-dibutyrate, substantially increases the extent of [32P]orthophosphate incorporation into the glycoprotein of normal and crushed nerve but not transected nerve. Biologically inactive 4 alpha-phorbol 12,13-didecanoate has no effect on P0 phosphorylation. Similarly, the addition of the cyclic AMP analog 8-bromo-cyclic AMP causes no appreciable changes in P0 labeling. These findings indicate that the phorbol ester-sensitive enzyme, protein kinase C, may be responsible for the phosphorylation of P0 within the myelin membrane.     

Taste systems of the petrosal ganglion of the rat glossopharyngeal nerve

Single unit recordings were taken from sensory ganglion cellsin the petrosal ganglion (PG) of the glossopharyngeal nerveof the rat. These taste units were examined with respect tospontaneous and evoked discharge patterns and responsivenessto a wide variety of chemical compounds, most of natural occurrence.Spontaneous activity patterns, with few exceptions, tended tobe extremely irregular with both bursting (clusters of 2–3spikes) and grouping (large groups of spikes as in evoked discharges).Most interspike interval histograms of spontaneous activitywere multimodal, similar to rat geniculate ganglion (GG) units.Evoked discharges usually displayed grouping of spikes, andlong latencies of onset and persistence of discharge after rinsewere sometimes seen. Little response was shown to nucleotidesor salts. Units responsive to amino acids tended to show largedischarge to only one or two amino acids; and the most responsiveamino acid usually varied from cell to cell. Units responsiveto alkaloids only responded to a few alkaloids with atropineand quinine being the most stimulatory. Units responsive toacids only discharged to a few of the acids tested and oftenacids of low pH elicited no discharge. Saccharin activated unitsresponsive to both sugar and alkaloids. A few units highly responsiveto both sugar and alkaloids were seen. The units were placedinto four clusters on the basis of chemicals activating themand certain neurophysiological characteristics: PG salt units,PG acid units and, tentatively, amino acid (sugar) units andX (alkaloid and alkaloid plus) units. The PG salt units didnot show the exclusive sensitivity to sodium and lithium compoundsas did the GG salt units. The PG acid units could also be differentiatedfrom the GG acid units. The petrosal amino acid and X units,on the other hand, could not be differentiated from similarunits in the rat GG.