Rhizobium meliloti exopolysaccharides: genetic analyses and symbiotic importance.

Genetic experiments have indicated that succinoglycan (EPS I), the acidic Calcofluor-binding exopolysaccharide, of the nitrogen-fixing bacterium Rhizobium meliloti strain Rm1021 is required for nodule invasion and possibly for later events in nodule development on alfalfa and other hosts. Fourteen exo loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. We have identified two loci, exoR and exoS, that are involved in the regulation of EPS I synthesis in the free-living state. Certain exo mutations which completely abolish EPS I production are lethal in an exoR95 or exoS96 background. Histochemical analyses of the expression of exo genes during nodulation using exo::TnphoA fusions have indicated that the exo genes are expressed most strongly in the invasion zone. In addition, we have discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for exopolysaccharides in symbiosis are discussed.     

Toxicity and moniliformin production by four recently described species of Fusarium and two uncertain taxa

Four recently described species, Fusarium nygamai, F. dlamini, F. beomiforme and F. napiforme and two uncertain taxa, F. nygamai from millet in Africa and Fusarium species from rice with Bakanae disease, were tested for toxicity and moniliformin production. Cultures grown on autoclaved corn were fed to groups of four one-day-old ducklings for 14 days. Isolates that caused the death of 3 or 4 out of 4 ducklings were considered to be toxic and analyzed for moniliformin. All 15 isolates of F. dlamini tested were nontoxic. The other taxa contained some isolates that were toxic to ducklings and produced moniliformin in corn cultures. This is the first report of moniliformin production by F. beomiforme (200–890 g/g), and F. napiforme (16–388 g/g), and by F. nygamai not obtained from millet in Africa (15–874 g/g). The highest production of moniliformin was obtained from the 19 isolates of F. nygamai from millet in Africa (4300–18200g/g) and the 15 isolates from rice with Bakanae disease (2300–19300 g/g). The taxonomic position of these two uncertain taxa should be re-evaluated.     

Regulation of axonemal Mg2+-ATPase from Paramecium cilia: effects of Ca2+ and cyclic nucleotides

Ciliary activity is regulated by Ca2+ and cyclic nucleotides, but the molecular mechanisms of the regulation are unknown. We have tested the ability of Ca2+ and cyclic nucleotides to alter ciliary Mg2+-ATPase or to stimulate phosphorylation of axonemal dynein. Mg2+-ATPase activity in cilia and axonemes from Paramecium was stimulated 2-fold by micromolar Ca2+, but this Ca2+ sensitivity was lost upon solubilization of the dyneins from the axoneme. The Ca2+-sensitive component of ciliary Mg2+-ATPase activity was inhibited by the dynein inhibitors vanadate and Zn2+, but was insensitive to the calmodulin antagonists calmidazolium and melittin. Dynein activity in the high-salt extract from axonemes was also insensitive to calmidazolium. Calmodulin did not sediment with 22 S or 12 S dyneins on sucrose gradients containing Ca2+, but it did sediment in the region from 19 S to 14 S. Mg2+-ATPase activity in ciliary fractions was unaltered in the presence of cAMP or cGMP. However, polypeptides associated with the 22 S and 12 S dyneins, as well as proteins of 19 S, 15 S, and 8 S, were substrates for endogenous ciliary kinases. High molecular weight polypeptides that sedimented at 22 S and 19 S were phosphorylated in a cyclic nucleotide-stimulated manner.     

Phospholamban forms Ca2+-selective channels in lipid bilayers

Phospholamban is the major membrane protein of the heart phosphorylated in response to beta-adrenergic stimulation. A role for phospholamban in the control of Ca2+ transport by the sarcoplasmic reticulum has been postulated, but the mechanism is incompletely understood. Structural characterization of the purified protein suggests that it is capable of forming a membrane-spanning pore (Simmerman, H. K. B., Collins, J. H., Theibert, J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261, 13333-13341). The experiments described here tested the hypothesis that canine cardiac phospholamban, isolated in the fully dephosphorylated state, forms ion channels in lipid bilayers. Phospholamban purified by two different methods formed channels that were permeable to cations, exhibited spontaneous openings and closings, and were selective for Ca2+ over K+. Dihydropyridine drugs and ryanodine did not affect channel activity. The putative membrane-spanning portion of the molecule, residues 26-52, also formed channels in the bilayer. The putative regulatory portion of the molecule, residues 2-25, did not. The results suggest that phospholamban may regulate sarcoplasmic reticulum Ca2+ flux by acting as a Ca2+ channel.     

Purification and properties of a vanadate- and N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit I (115 kDa) of the H+-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 microM N-ethylmaleimide or 5 microM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na+/K+-ATPase and the gastric K+/H+-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [alpha-32P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg2+ and strongly inhibited by Ca2+ ions. Similarly, the ATPase activity was dependent on Mg2+ and inhibited by the presence of Ca2+ ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F-, which inhibited the vanadate-sensitive ATPase. Incubation of the enzyme in the presence of [14C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [alpha-32P]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.     

Prolongation and cessation of estrous cycles in aging C57BL/6J mice are differentially regulated events

The relative contributions of ovarian failure and hypothalamic-pituitary dysfunction to the prolongation and cessation of estrous cycles were assessed by measuring the ability of acutely ovariectomized (OVX) middle-aged (12 mo) mice to cycle after receiving grafts (under the renal capsule) of ovaries from young (2 mo) mice. The potentially disruptive effect of the acyclic state on the cycling response to grafted, young ovaries was avoided restricting grafting to middle-aged hosts that were still cycling. The effect of chronic exposure to ovarian secretions before the cessation of cyclicity on age-related hypothalamic-pituitary dysfunction was also assessed. The cycling ability of long-term OVX middle-aged mice (i.e., OVX at 3 mo) bearing grafts of young ovaries was compared to that of age-matched acutely OVX controls. Grafted young ovaries extended the cycling lifespan of acutely OVX middle-aged hosts by 60%. The length of this extended cycling lifespan, however, was only 80% of that achieved by young hosts bearing grafts of young ovaries. Young ovaries in middle-aged mice markedly lowered the incidence of long cycles (greater than 5 days), shifting the modal cycle length to 5 days. However, young ovaries in middle-aged mice failed to increase the incidence of 4-day cycles, the modal cycle of young controls. Middle-aged ovaries grafted into young hosts lengthened their cycles and shortened their cycling lifespan to middle-aged values. Long-term ovariectomy failed to increase the cycling lifespan of middle-aged hosts bearing grafts of young ovaries beyond that achieved in acutely OVX mice. Long-term ovariectomy did shorten the modal cycle length of middle-aged mice to 4 days, although the duration of 4-day cycling was only one-third (2 mo) that of young controls. These results indicate that the relative contributions of ovarian and neuroendocrine factors to three major events of reproductive aging vary with each event. Whereas the hypothalamic-pituitary unit appears to play an important role in the initial shift from 4- to 5-day cycles, the aging ovary plays the major role in the subsequent shift to longer cycles and in the ultimate cessation of cyclicity. Although chronic exposure to ovarian secretions during the period of cyclicity does not play a major role in the cessation of cyclicity, it appears to contribute to the hypothalamic-pituitary changes responsible for the initial shift from 4- to 5-day cycles.     

Inhibitory Effect of Autoclaving Whey-Based Medium on Propionic Acid Production by Propionibacterium shermanii

Propionic acid production by Propionibacterium shermanii was compared in pasteurized and autoclaved whey-based media. Propionic acid production decreased with increasing whey concentration in autoclaved media but not in pasteurized media. Increasing the yeast extract concentration from 5 to 10 g/liter greatly reduced the inhibitory effect of autoclaving.     

Phosphorylation of the mouse hepatitis virus nucleocapsid protein

Analysis of the radiolabeled tryptic peptides derived from the nucleocapsid proteins of two serotypes of mouse hepatitis virus showed each to have a small number of unique peptides; however, two biologically distinct variants of the JHM strain appeared identical. Analysis of [32P]-labeled nucleocapsid-derived peptides showed that phosphorylation occurs at only a few sites and that all three viruses differed in the sites of phosphorylation. No differences in the sites of phosphorylation were found between the nucleocapsid proteins derived from purified virions and the membranes or the cytosol of infected cells, suggesting that post-translational phosphorylation plays no role in the regulation of viral assembly. These data show unequivocal evidence that the nucleocapsid proteins of mouse hepatitis virus strains differ in the sites of phosphorylation.     

The effect of thioridazine on haloperidol induced behavioral hypersensitivity

Behavioral Hypersensitivity (BH) to dopamine agonists occurs following chronic treatment with most neuroleptics including haloperidol. In the present study we observed that the concurrent administration of thioridazine and haloperidol prevented the development of BH. In contrast, another neuroleptic, fluphenazine, coadministered with haloperidol, potentiated the degree of BH relative to animals treated with haloperidol only. In rats already made hypersensitive by chronic treatment with haloperidol, a 4 week subsequent treatment with normal saline, thioridazine alone of thioridazine in combination with haloperidol, produced normal behavioral responsiveness. These results suggest that thioridazine prevents the development of BH and can reverse the expression of haloperidol-induced BH.     

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Summary Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CF_o-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A⁺-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.