Relationships within theRugopharynx delta species complex (Nematoda: Strongyloidea) from Australian marsupials inferred from allozyme electrophoretic data

Relationships between the strongyloid nematodesRugopharynx delta, R. zeta, R. omega, R. longibursaris, R. mawsonae andR. sigma, all from macropodid marsupials, were investigated using allozyme data. The phylogenetic trees derived from the electrophoretic data set were congruent with those of the hosts and were consistent with the hypothesis that the species complex originated in pademelons of the genusThylogale and diversified in rock-wallabies (Petrogale spp.) and scrub wallabies of the subgenusNotamacropus. Host switching is evident only between closely related macropodid taxa.     

Mating behaviour and parapatry in two species of Australian reptile tick

Summary Few quantitative studies have examined the ecological consequences of similarities and/or differences in mating behaviour of parapatric species. Reproductive interference occurs between several parapatric species of Australian reptile tick, due to similarities in their mating behaviour (Andrews et al. 1982a). Attempts to determine whether reproductive interference serves to maintain parapatry between Amblyomma limbatum and Aponomma hydrosauri have been hindered because of difficulties in providing conditions conducive to conspecific mating in Amb. limbatum. The present study examined whether off-host and/or onhost temperature influenced the subsequent mating behaviour (i.e. the proportion of females that mate and the time when mating occurs) of these two species. Irrespective of the temperature experienced by ticks prior to host attachment, specific on-host temperatures were needed to induce mating in Amb. limbatum (i.e. host cloacal temperatures >32° C prior to the time of peak mating activity). Significantly more Amb. limbatum females were mated and the time taken by females to mate decreased with increasing on-host temperatures. mating in Ap. hydrosauri occurred over a wider range of on-host temperatures and the time when mating occurred did not alter at different on-host temperatures. In addition, significantly more Ap. hydrosauri males moved and each male made more moves on hosts than did Amb. limbatum males. It is suggested that Ap. hydrosauri may in consequence have a competitive mating advantage over Amb. limbatum at a boundary. Similarities in mating behaviour, on the other hand, increase the probability of reproductive interference, hence reduce the reproductive fitness of colonizing females of both species. We propose that similarities and differences in mating behaviour could play a critical role in the maintenance of parapatric boundaries.     

T-DNA and opine synthetic loci in tumors incited by Agrobacterium tumefaciens A281 on soybean and alfalfa plants.

We report here the molecular characterization of transferred DNA (T-DNA) in leguminous tumors incited by Agrobacterium tumefaciens A281 harboring the tumor-inducing plasmid pTiBo542. The T-DNA is composed of two regions named TL (left portion)-DNA and TR (right portion)-DNA, in accordance with the nomenclature for the octopine strains. TL-DNA is defined by several internal HindIII restriction fragments totaling 10.8 kilobase pairs (kbp) in uncloned soybean and alfalfa tumors. Alfalfa tumor DNA may contain one more HindIII fragment at the left end of TL-DNA than does soybean tumor DNA. TR-DNA has a 5.8-kbp BamHI-EcoRI internal fragment. All borders other than the left border of TL-DNA appear to be the same within the detection limits of Southern blot hybridization experiments. The two T-DNA regions are separated by 16 to 19 kbp of DNA not stably maintained in tumors. The distance from the left border of TL-DNA to the right border of TR-DNA is approximately 40 kbp. Loci for the mannityl opines are situated in TR-DNA, based on genetic and biochemical criteria.     

Genetic evidence for a complex of species within Rugopharynx australis (Mönnig, 1926) (Nematoda: Stronglyloidea) from macropodid marsupials

An electrophoretic analysis using 17 enzyme loci was carried out on specimens of the gastric nematode of macropodid marsupials, Rugopharynx australis (Mönnig, 1926), collected from Macropus eugenii (Desmarest), M. fuliginosus (Desmarest), M. giganteus Shaw, M. robustus Gould, M. rufogriseus (Desmarest), M. rufus (Desmarest), Thylogale billardierii (Desmarest) and Wallabia bicolor (Desmarest) from south-eastern Australia. The extent of fixed genetic differences between nematodes from different host species ranged from 0–53%. The two distinct morphological forms of the parasite found in M. rufogriseus differed at 50% of loci. Specimens present in M. fuliginosus and M. giganteus were indistinguishable genetically, as were nematodes from M. rufus and M. robustus. Of the two morphologically distinct congeners included in the analysis as controls, Rugopharynx epsilon (Johnston & Mawson, 1939) was genetically distinct (46–69% fixed genetic differences) from all specimens of the R. australis complex while R. rufogrisea Magzoub, 1964 was closely related to one of the two species occuring in M. rufogriseus. It was concluded that R. australis is a species complex, with a genetically distinct species present in M. eugenii, M. fuliginosus/M. giganteus, M. robustus/M. rufus, W. bicolor and T. billardierii, and two species in M. rufogriseus.     

In vitro transformation of petunia cells by an improved method of co-cultivation with A. tumefaciens strains

Summary A method (termed co-cultivation) for transforming plant cells in vitro with A. tumefaciens strains, which was originally developed by Marton et al. (1978) Nature 277: 129–131, has been modified by the incorporation of a novel feeder plate culture system and been extended to use with petunia protoplasts. Using efficient cell plating and selection conditions for phytohormone-independent growth, large numbers of independent transformed calli can be obtained efficiently (10^-1) and in less than 3 weeks following protoplast isolation. Southern hybridization analysis has confirmed that the majority of the resulting in vitro transformants contain a single copy of full length T-DNA.The high efficiency of this procedure allows simple screening to identify plant cells transformed by Ti plasmids attenuated by deletion of internal T-DNA regions. Results are presented that demonstrate the co-cultivation method can be used in conjunction with short term assays for monitoring plant gene expression.     

Platelet activating factor. Stimulation of the lipoxygenase pathway in polymorphonuclear leukocytes by 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine

1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (AAGPC) triggered the release of [3H]arachidonate but not [14C]stearate from cellular phospholipids in cytochalasin B-treated rabbit polymorphonuclear leukocytes. Concentrations of AAGPC up to 20 nM caused a dose-dependent release and subsequent metabolism of the released [3H]arachidonic acid. Most of the release of the [3H]arachidonate had taken place within the first 2 min of stimulation. Phosphatidylinositol and phosphatidylcholine served as the sources of [3H]arachidonate with about 50% of the label coming from each pool. Challenge of cytochalasin B-treated polymorphonuclear leukocytes with AAPGC led to the production of [3H]hydroxyeicosatetraenoic acids and [3H]dihydroxyeicosatetraenoic acids. No significant production of [3H]prostaglandins or [3H]thromboxanes was detected. AAGPC also caused a dose-dependent degranulation of cytochalasin B-treated rabbit polymorphonuclear leukocytes as shown by the release of beta-glucuronidase and lysozyme. Both the AAGPC-stimulated production of arachidonate metabolites and the degranulation response were blocked by eicosatetraynoic acid and non-dihydroguaiaretic acid at similar inhibitor concentrations. These findings suggest the bioactions of AAGPC on polymorphonuclear leukocytes may be mediated by the release of arachidonic acid and the production of mono- and dihydroxyeicosatetraenoic acids.     

Spontaneous mutation conferring the ability to catabolize mannopine in Agrobacterium tumefaciens.

Two nopaline-type strains of Agrobacterium tumefaciens, C58 and T37, as well as strain A136, which is a Ti plasmid-cured derivative of strain C58, gave rise to spontaneous mutants that were able to grow on mannopine. The observation of mutagenesis with strain A136 demonstrated that the ability to acquire this new catabolic potential was independent of the presence of a Ti plasmid. The mutants were isolated after 4 weeks of incubation on minimal medium containing mannopine as the sole carbon source. They also utilized mannopinic acid, but not agropine or agropinic acid. In addition, the spontaneous mutant LM136, but not its parent strain A136, degraded many mannityl opine analogs. [14C]mannopine disappeared in the presence of LM136 cells which had been pregrown on opine or nonopine substrates. These results suggested that the catabolic system of this mutant was not subject to a stringent regulation. A clone conferring the ability to utilize mannopine on a recipient pseudomonad was selected from a genomic library from both the mutant LM136 and its parent strain. Only the LM136 clone was expressed in the parent Agrobacterium strain A136. Southern analysis showed that the genes for mannopine catabolism in the spontaneous mutants differed from the corresponding Ti plasmid-encoded genes of octopine-type or agropine-type Agrobacterium strains. Cells of LM136 utilized [14C]mannopine without generating detectable amounts of intracellular agropine. In contrast, a major fraction of the radioactivity recovered from cells of the octopine-type strain Ach5, after incubation on [14C]mannopine, was in the form of agropine.     

Lipopolysaccharide priming of alveolar macrophages for enhanced synthesis of prostanoids involves induction of a novel prostaglandin H synthase.

We report here that lipopolysaccharide (LPS) priming of rabbit alveolar macrophages leads to amplified synthesis of prostanoids, at least in part, by induction of a novel prostaglandin H synthase (PGH synthase). Rabbit alveolar macrophages were cultured with or without added LPS derived from Escherichia coli 0111:B4 for 4 h and then stimulated with opsonized zymosan (OPZ). LPS priming of alveolar macrophages resulted in enhanced release of thromboxane (TX) upon stimulation with OPZ, when compared to stimulated non-LPS controls. Addition of exogenous arachidonic acid to LPS-primed alveolar macrophages also resulted in increased production of TX. The LPS-induced increase in TX formation, in response to OPZ or arachidonic acid, was abolished by the addition of actinomycin D or cycloheximide during the priming period. Gas chromatography/mass spectrometry analysis indicated that levels of prostaglandins D2, E2, and F2 alpha, along with TX, were augmented in stimulated LPS-primed alveolar macrophages, implicating PGH synthase in the priming process. PGH synthase enzymatic activity, as determined by addition of arachidonic acid to macrophage sonicates, was markedly enhanced in LPS-primed alveolar macrophages. This correlated with increased PGH synthase levels detected by immunoprecipitation of 35S-labeled proteins and by Western blot analysis. Finally, Northern blot analysis using a cDNA probe to the recently described mitogen-inducible mouse PGH synthase revealed strong induction of approximately 4.3-kilobase mRNA in LPS-primed alveolar macrophages. Taken together, these results reveal that induction of a novel PGH synthase, probably the rabbit homologue of PGH synthase-2, plays a role in the enhanced synthesis of prostanoids by LPS-primed alveolar macrophages.     

Inhibition by adenosine 3':5'-monophosphate of eicosanoid and platelet-activating factor biosynthesis in the mouse PT-18 mast cell.

A mouse spleen-derived mast cell line (PT-18) was employed to examine the mechanisms of adenosine 3':5'-monophosphate (cAMP)-mediated inhibition of antigen-induced lipid mediator biosynthesis. Specifically, we tested the hypothesis that increasing cAMP in mast cells inhibits lipid mediator biosynthesis by a mechanism independent of effects on histamine release (degranulation) or changes in cytosolic calcium concentration. Forskolin inhibited antigen-induced prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and leukotriene B4 (LTB4) production by 30-50%. In contrast, forskolin had no inhibitory effect on antigen-induced increases in cytosolic calcium concentration, as monitored by the calcium indicator fura-2, or histamine release from the cells. The combination of the phosphodiesterase inhibitor isobutylmethylxanthine with forskolin inhibited the antigen-induced production of PGD2 and LTC4 by 90-100% and histamine release by about 60%. These responses were accompanied by a virtual abolition of the antigen-induced increase in cytosolic calcium. To test further the hypothesis that increasing cAMP can lead to inhibition of lipid mediator biosynthesis in the absence of effects on cytosolic calcium, we employed the calcium ionophores A23187 and ionomycin. Forskolin alone or in combination with isobutylmethylxanthine had no effect on ionophore-induced increases in cytosolic calcium but effectively inhibited leukotriene biosynthesis. In addition, increasing cyclic AMP led to an inhibition of ionophore-induced production of platelet-activating factor and liberation of arachidonic acid. These data suggest that a relatively modest increase in cAMP-dependent protein kinase activity in mast cells leads to inhibition of the lipase-catalyzed cleavage of arachidonic acid from membrane phospholipids in the absence of measurable effects on either histamine release or changes in cytosolic calcium concentration. This effect results in a selective inhibition of the biosynthesis of lipid mediators including LTC4, LTB4, PGD2, and platelet-activating factor.     

Fungal Catabolism of Crown Gall Opines

This study was conducted to determine the capacities of 37 fungi to utilize various crown gall opines as their sole carbon and nitrogen source. One strain of Fusarium solani, two of Cylindrocarpon destructans, and six of Cylindrocarpon heteronema catabolized octopine, mannopine, octopinic acid, succinamopine, or a combination of these opines. One C. heteronema and one Fusarium dimerum strain grew only on succinamopine. None of the fungal isolates had the ability to grow on nopaline. The catabolism of opines by fungi was confirmed by the disappearance of the opine from the growth medium and by an increase in final mycelial dry weight with rising initial concentration of test substrate. This study thus shows that the catabolism of opines is not restricted to bacteria.