The Pyrophosphate-Dependent Phosphofructokinase of the Protist, Trichomonas vaginalis, and the Evolutionary Relationships of Protist Phosphofructokinases

The pyrophosphate-dependent phosphofructokinase (PP_i-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5′ half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PP_i-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PP_i-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PP_i-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the α- and β-subunits of plant PP_i-PFKs. The third group (``X') containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group (``Y') comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PP_i-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers. Received: 5 December 1997 / Accepted: 18 March 1998     

Uptake and translocation of phosphate by pho2 mutant and wild-type seedlings of Arabidopsis thaliana

The pho2 mutant of Arabidopsis thaliana (L.) Heynh. accumulates excessive Pi (inorganic phosphate) concentrations in shoots compared to wild-type plants (E. Delhaize and P. Randall, 1995, Plant Physiol. 107: 207–213). In this study, a series of experiments was conducted to compare the uptake and translocation of Pi by pho2 with that of wild-type plants. The pho2 mutants had about a twofold greater Pi uptake rate than wild-type plants under P-sufficient conditions and a greater proportion of the Pi taken up accumulated in shoots of pho2. When shoots were removed, the uptake rate by roots was found to be similar for both genotypes, suggesting that the greater Pi uptake by the intact pho2 mutant is due to a greater shoot sink for Pi. Although pho2 mutants could recycle ³²Pi from shoots to roots through phloem the proportion of ³²Pi translocated to roots was less than half of that found in wild-type plants. When transferred from P-sufficient to P-deficient solutions, Pi concentrations in pho2 roots had a similar depletion rate to wild-type roots despite pho2 shoots having a fourfold greater Pi concentration than wild-type shoots throughout the experiment. We suggest that the pho2 phenotype could result from a partial defect in Pi transport in the phloem between shoots and roots or from an inability of shoot cells to regulate internal Pi concentrations. Received: 20 August 1997 / Accepted: 4 October 1997     

Towards European climate risk surfaces: the extent and distribution of analogous and non-analogous climates 1931–2100

Aim Climate is an important determinant of species distributions. We assess different aspects of risk arising from future climate change by quantifying changes in the spatial distribution of future climatic conditions compared with the recent past. Location Europe. Methods A 10′ × 10′ resolution gridded data set of five climate variables was used to calculate expected changes to the area, distance and direction of 1931–60 climatic conditions under the HadCM3 climate model for four future climate scenarios based on different rates of greenhouse gas emissions (SRES scenarios). Three levels of tolerance ranges determined the thresholds for which future conditions are considered analogous to 1931–60 (pre‐warming) conditions. Results For many parts of Europe, areas with pre‐warming analogous climate conditions will be smaller and further away in the future than they are now. For any location in Europe, areas with pre‐warming analogous mean annual temperature conditions will, on average, be reduced between 23.7% (B1 scenario) and 49.7% (A1FI scenario) by 2100 when assuming a medium tolerance range. The mean distance to these areas will, on average, increase between 272 km (B1) and 645 km (A1FI). These changes are more pronounced for temperature than for water availability variables and also for narrow tolerance ranges compared to wide tolerance ranges. Using a combined measure of both temperature and precipitation variables, areas with prewarming analogous conditions are predicted to be in a more northeasterly direction in the future, but there are considerable regional differences within Europe. Main conclusions The results suggest that, for some parts of Europe, the loss of area with any suitable climatic conditions represents the greatest risk to biodiversity, but in other regions the distances that species may have to move to reach suitable climatic conditions may be a greater problem. Quantifying the distance and direction in analyses of change of climatically suitable areas can add additional information for climate change risk assessments.     

Specific Detection of Xanthomonas axonopodis pv. dieffenbachiae in Anthurium (Anthurium andreanum) Tissues by Nested PCR

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (10³ CFU ml⁻¹) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.     

What is a Suiforme (Artiodactyla)?: Contribution of Cranioskeletal and Mitochondrial DNA Data

Suiformes (Artiodactyla) traditionally includes three families: Suidae, Tayassuidae, and Hippopotamidae but the monophyly of this suborder has recently been questioned from molecular data. A maximum parsimony analysis of molecular, morphological, and combined data was performed on the same set of taxa including representatives of the three Artiodactyla suborders (Suiformes, Ruminantia, and Tylopoda) and Perissodactyla as outgroup. Mitochondrial (cytochromeband 12S rRNA) sequence comparisons support the monophyly of Suina (Suidae and Tayassuidae) and Ancodonta (Hippopotamidae) but not the monophyly of Suiformes. Inversely, our preliminary morphological analysis supports the monophyly of Suiformes whereas relationships among the three families are not resolved. The combined data set does not resolve the relationships between Suina, Ancodonta, and Ruminantia. These results are discussed in relation to morphological characters and paleontological data. Some improvements are suggested to clarify the morphological definition of Suiformes and relationships among them.     

Electron spin resonance detection of extracellular superoxide anion released by cultured endothelial cells

Objective and Methods Endothelium produces oxygen-derived free radicals which play a major role in vessel wall physiology and pathology. Whereas NO^· production from endothelium has been extensively characterized, little is known about endothelium-derived O^-·₂. In the present study, we determined the O^-·₂ production of bovine aortic endothelial cells (BAEC) using the spin trap 5,5-dimethyl-1 pyrroline-N-oxide (DMPO) and electron spin resonance (ESR) spectroscopy.

Results An ESR adduct DMPO-OH detected in the supernatant of BAEC after stimulation with the calcium ionophore A23187 originated from the trapping of extracellular O^-·₂, because coincubation with superoxide dismutase (30 U/ml) completely suppressed the ESR signal, whereas catalase (2000 U/ml) had no effect. A23187 stimulated extracellular O^-·₂ production in a time- and dose-dependent manner. The coenzymes NADH and NADPH both increased the ESR signal, whereas a flavin antagonist, diphenylene iodonium, abolished the ESR signal. Phorbol myristate acetate potentiated, whereas bisindolylmaleimide I inhibited the A23187-stimulated O^-·₂ production, suggesting the involvement of protein kinase C. These signals were not altered L-NAME, a NO-synthase inhibitor, suggesting that the endogenous production of NO^· did not alter O^-·₂ production. Finally, the amount of O^-·₂ generated by A23187-stimulated post-confluent BAEC was one order of magnitude higher than that evoked by rat aortic smooth muscle cells stimulated under the same conditions.     

Vessel-diameter distribution in roots of grapevines (Vitis vinifera L. cv. Shiraz)

The distribution frequency patterns of diameter of xylem vessels and percentage of total predicted axial conductances were studied in 190-day and 212-day-old main roots of grapevine (Vitis vinifera L. cv. Shiraz) grown under well-watered and stressed conditions. The protoxylem were the first to mature and were responsible for most of the theoretical conductance in root segments between the tip and 2.5 cm from the tip. Some large xylem vessels retained cross walls and protoplasm up to 22.5 cm from the tip. Statistical tests using the Kolmogorov-Smirnov two sample test showed that the pattern of distribution frequency of xylem vessels classified in different diameter classes varied with distance from the root tip. The distribution frequency of xylem vessels was similar in both well-watered and stressed plants from the tip up to 15 cm from the tip. At distances further from the tip the distribution frequency of xylem vessels of well-watered plants was significantly different from that of stressed plants, with the former having more larger vessels than the latter. The pattern of vessel distribution frequency was different from that of percent total axial conductance (K_h) predicted with fewer large vessels carrying most of the axial flow.     

Studies on conversion of labelled pregnenolone to progesterone by olive baboon placental cytotrophoblast cells in vitro

The experiments were designed to study the influence of a variety of substances, which have been reported to affect placental steroid metabolism, on pregnenolone metabolism by olive baboon placenta cells. Placentae were obtained from six baboons by caesarian section between 100 and 110 days of pregnancy. The cells were isolated by enzyme digestion and Ficoll gradient separation and incubated in Ham's F10 media with 5μ Ci [4,7, — 3H] pregnenolone in the presence or absence of indomethacin (0.1 mmol/1), dibutyryl cAMP (10 mmol/l), phorbol myristic acetate (10 nmol/l), 3-isobutyl-1-methylaxanthine (0.5 mmol/l), calcium ionophore A23187 (1 μmol/l), and nordihydroguaiaretic acid (40 μg/ml). Control experiments were done using leucocytes, inactivated placental cells and Ham's F10 media. Time course and dose response studies were also done. Placental cells converted pregnenolone to progesterone in a dose related manner. Addition of test compounds did not affect conversion rates. It is concluded that further studies are needed for elucidation of mechanisms that regulate progesterone synthesis in baboon placentae.