Recombination-deficient mutants of Salmonella typhimurium are avirulent and sensitive to the oxidative burst of macrophages

Mutations in the genes recA and recBC were constructed in the virulent Salmonella typhimurium strain 14028s. Both the recA and recBC mutants were attenuated in mice. The mutants were also sensitive to killing by macrophages in vitro. The recombination mutants were no longer macrophage sensitive in a variant line of J774 macrophage-like cells that fail to generate superoxide. This suggests that repair of DNA damage by Salmonella is necessary for full virulence in vivo and that the oxidative burst of phagocytes is one source of such DNA damage.     

MITOCHONDRIAL-DNA ANALYSES AND THE ORIGIN AND RELATIVE AGE OF PARTHENOGENETIC LIZARDS (GENUS CNEMIDOPHORUS). III. C. VELOX AND C. EXSANGUIS

Mitochondrial DNAs (mtDNAs) of two unisexual, parthenogenetically reproducing species of whiptail lizards (Cnemidophorus velox and C. exsanguis) and their bisexual relatives were compared by restriction-enzyme analysis to assess levels of mtDNA variation and to establish the maternal ancestry of the unisexuals. No cleavage-site differences were found to be diagnostic between C. velox and C. exsanguis mtDNAs, suggesting an ancestry rooted in the same maternal lineage. The mtDNA of the unisexuals is relatively homogeneous, indicating that these lineages are of recent origin. Phylogenetic analysis revealed that the maternal ancestor of both C. velox and C. exsanguis was most probably C. burti stictogrammus, C. costatus barrancorum, or an unidentified taxon closely related to them. In addition, the mtDNA analyses demonstrate conclusively that the triploid species C. velox could not have been formed by the fertilization of an unreduced (diploid) C. inornatus egg, further strengthening the hypothesis that parthenogenesis in Cnemidophorus results from hybridization.     

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.     

Managing the pattern of forest harvest: lessons from wildfire

Managing forests for sustainable use requires that both the biological diversity of the forests and a viable forest industry be maintained. A current approach towards maintaining biological diversity is to pattern forest management practices after those of natural disturbance events. This paradigm hypothesizes that ecological processes will be maintained best where active management approximates natural disturbance events. The forest management model now used in most sub-boreal and boreal forests calls for regularly dispersed clearcuts no greater than 60–100 ha in size. However, the spatial characteristics of the landscape produced by this model are distinctly different from the historic pattern generated by wildfire, which was heretofore the dominant stand-replacing process in these forests. Wildfire creates a more complex landscape spatial pattern with greater range in patch size and more irregular disturbance boundaries. Individual wildfires are often over 500 ha but leave patches of unburned forest within them. The combination of these attributes is not present in recent clearcuts. Allowing a proportion of larger (i.e.>500ha) harvest units may provide distinct economic advantages that could outweight the opportunity costs of leaving some patches of forest behind. For the forest type examined, further evaluation of modelling forest harvest patterns more closely after the patterns created by wildfire is required as it may achieve a good balance and strike a suitable compromise between certain ecological and economic objectives of sustainable development.     

Evaluation of a remotely operated vehicle (ROV) as a tool for studying the distribution and abundance of zooplankton

Conventional methods for measuring zooplankton distributionsare too laborious and time consuming to permit sufficient temporaland spatial resolution in many instances. An ability to makemore efficient and precise measurements would be useful. Weevaluated the potential for using the video system of a commerciallyavailable remotely operated vehicle (ROV) to measure the distributionand abundance of zooplankton by calibrating ROV counts withcounts based on a conventional sampling procedure (a Schindlertrap), and by using an ROV to measure the density of zooplanktonin a small lake. As configured here, this particular ROV wassuitable for measuring the density of the cladocerans Daphniaand Holopedium. It was also suitable for assessing the distribution,but not absolute densities, of Chaoborus and Leptodora. Imagequality was inadequate for quantitative estimates of copepod(Diaptomus minutus) abundance, and prevented us from studyingbehavioral responses of copepods to the vehicle. We concludethat the ROV has at least three useful features: it can be usedto locate patches of those species that are imaged effectively;a large number of samples (videotapes) can be collected almostsynoptically with high spatial resolution; the ROV enables insitu observation of zooplankton. The ROV also has three importantlimitations: the small image volume makes it difficult to studyrare organisms; inadequate image resolution precludes studiesof relatively small organisms (e.g. the calanoid copepod D.minutus);zooplankton respond to the presence of the ROV.     

THE IMPACT OF TREE SQUIRRELS (TAMIASCIURUS) ON LIMBER PINE SEED DISPERSAL ADAPTATIONS

Tree squirrels (Tamiasciurus) are important selective agents on conifer reproductive strategies (Smith 1970, 1975). Although this is well established for wind-dispersed pines, the impact of tree squirrels on bird-dispersed pines has been largely ignored. I assessed the impact of tree squirrels on the allocation of reproductive energy in the bird-dispersed limber pine (Pinus flexilis) by comparing its cone and seed traits from three sites in the Rocky Mountains where tree squirrels (Tamiasciurus) are present to those from three mountain ranges in the Great Basin where tree squirrels are absent. As predicted, differences between the two regions in individual cone and seed traits are consistent with the hypothesis that tree squirrels are important selective agents on these traits. In the absence of tree squirrels, limber pine allocates more than twice as much energy to kernel compared with that invested in putative seed defenses (cone, resin, and seed coat) as does limber pine where tree squirrels are present. Such a large difference is particularly striking, because tree squirrels may have become extinct in the Great Basin in only the last 12,000 yr. Although many factors influence the allocation of energy to cones and seeds, no single factor other than the presence of tree squirrels is compatible with the large and consistent differences between limber pine in the Rocky Mountains and Great Basin. These results show that tree squirrels are an important constraint on the evolution of cone and seed traits that promote the dispersal of seeds by birds.     

The transposable element mariner can excise in non-drosophilid insects

Plasmid-based excision assays performed in embryos of two non-drosophilid species using the mariner transposable element from Drosophila mauritiana resulted in empty excision sites identical to those observed after the excision of mariner from D. mauritiana chromosomes. In the presence of the autonomous mariner element Mos1, excision products were recovered from D. melanogaster, D. mauritiana and the blowfly Lucilia cuprina. When a hsp82 heat shock promoter-Mos1 construct was used to supply mariner transposase, excision products were also recovered from the Queensland fruitfly Bactrocera tryoni. Analysis of DNA sequences at empty excision sites led us to hypothesise that the mariner excision/repair process involves the formation of a heteroduplex at the excision breakpoint. The success of these assays suggests that they will provide a valuable tool for assessing the ability of mariner and mariner-like elements to function in non-drosophilid insects and for investigating the basic mechanisms of mariner excision and repair.     

Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.     

A role for ornithine in the regulation of putrescine accumulation and ornithine decarboxylase activity in Reuber H35 hepatoma cells

We investigated the ability of intracellular ornithine to alter both the biosynthesis of putrescine and the activity of ornithine decarboxylase in Reuber H35 hepatoma cells in culture incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA). In confluent cultures of H35 cells, the addition of TPA (1.6 μM) caused the activity of ornithine decarboxylase to increase by more than 100-fold within 4 h. When exogenous ornithine (0.1–1.0 mM) was added to the culture medium with TPA, a marked dose-dependent increase in the production of putrescine was observed. The activity of ornithine decarboxylase in the same cultures incubated with ornithine decreased in a similar dose-dependent manner. The addition of arginine (0.1–1.0 mM) (but not lysine or histidine) to the H35 cells in culture concomitant with TPA also led to a relative increase in putrescine biosynthesis and a decrease in ornithine decarboxylase activity compared to cultures not receiving the amino acids. A similar response to exogenous ornithine and TPA was observed in a series of less confluent rapidly growing cultures which were in culture for a shorter period of time. The confluent cultures possessed a basal level of arginase (55 units/mg protein) which increased approx. 2-fold upon treatment with TPA. The intracellular concentration of ornithine in the unstimulated cells was in the order of 0.02–0.03 mM. Upon incubation of the cells with exogenous ornithine or arginine, the intracellular pools of these amino acids increased 4- to 8-fold.     

Nucleotide sequence determination of guinea-pig casein B mRNA reveals homology with bovine and rat alpha s1 caseins and conservation of the non-coding regions of the mRNA.

Nucleotide sequence analysis of cloned guinea-pig casein B cDNA sequences has identified two casein B variants related to the bovine and rat alpha s1 caseins. Amino acid homology was largely confined to the known bovine or predicted rat phosphorylation sites and within the 'signal' precursor sequence. Comparison of the deduced nucleotide sequence of the guinea-pig and rat alpha s1 casein mRNA species showed greater sequence conservation in the non-coding than in the coding regions, suggesting a functional and possibly regulatory role for the non-coding regions of casein mRNA. The results provide insight into the evolution of the casein genes, and raise questions as to the role of conserved nucleotide sequences within the non-coding regions of mRNA species.