Ecological and anatomical characterization of some Pinus patula ectomycorrhizas from Mpumalanga, South Africa

 Ectomycorrhizal (ECM) fungi are an important component of the Pinus patula Schlechdt. et Cham. forest ecosystem in Mpumalanga, South Africa. ECM roots are intimately associated with accumulated litter on the forest floor and four ECM isolates where examined to determine whether they provide plant access to inorganic and organic sources of nitrogen (N) and inorganic, complexed inorganic and organic sources of phosphorus (P). In in vitro studies, all isolates were found to utilize most of the organic N compounds and organic or complexed inorganic P compounds supplied. Thus, ECM fungi could play a significant role in providing N and P to P. patula, especially from sources to which the host plant would not normally have access. Temperature sensitivities and pH optima of the four isolates differed. Of the ECM isolates WITS 01 and WITS 06 were collected from a high-litter site; WITS 01 mycorrhizas, identified as Scleroderma citrinum, were white, smooth and dichotomously branched with smooth, pale yellow, differentiated rhizomorphs. The mantle was plectenchymatous with outer and inner layers showing ring-like arrangements of hyphal bundles. The Hartig net had a palmetti shape. The WITS 02 (not identified) mycorrhizas were brown with lighter coloured root tips, with simple to dichotomous branching, smooth with no distinct mantle and sparse hyphae occurred on the root surface. The Hartig net was palmetti type with lobed haustoria. The results are discussed in relation to ECM distribution and function in nutrient cycling. Accepted: 7 January 1999     

Endocrine Profiles, Haematology and Pregnancy Outcomes of Late Pregnant Holstein Dairy Heifers Sired by Bulls Giving a High or Low Incidence of Stillbirth

The high incidence of stillbirth in Swedish Holstein heifers has increased continuously during the last 15 years to an average of 11% today. The pathological reasons behind the increased incidence of stillbirth are unknown. The present experiment was undertaken to investigate possible causes of stillbirth and to study possible physiological markers for predicting stillbirth. Twenty Swedish Holstein dairy heifers sired by bulls with breeding values for a high risk of stillbirth (n = 12) (experimental group) and a low risk of stillbirth (n = 8) (control group, group B) were selected based on information in the Swedish AI-data base. The experimental group consisted of 2 subgroups of heifers (groups A1 and A2) inseminated with 2 different bulls with 3.5% and 9% higher stillbirth rates than the average, and the control group consisted of heifers pregnant with 5 different bulls with 0%–6% lower stillbirth rates than the average. The bull used for group A1 had also calving difficulties due to large calves as compared to the bull in group A2 showing no calving difficulties. The heifers were supervised from 6–7 months of pregnancy up to birth, and the pregnancies and parturitions were compared between groups regarding hormonal levels, haematology, placental characteristics and calf viability. In group A1, 1 stillborn, 1 weak and 4 normal calves were recorded. In group A2, 2 stillborn and 4 normal calves were registered. All animals in the control group gave birth to a normal living calf without any assistance. The weak calf showed deviating profiles of body temperature, saturated oxygen and heart rates, compared with the normal living calves. No differences of the placentome thickness, measured in vivo by ultrasonography were seen between the groups. The number of leukocytes and differential cell counts in groups A1 and A2 followed the profiles found in the control group. In group A1, a slight decrease of oestrone sulphate (E1SO4) levels was found in the animal delivering a stillborn calf from the first 24-h blood sampling at 6 weeks to the second at 3 weeks prior to delivery, while the levels of E1SO4 at both periods in the animal delivering a weak calf followed the profile in animals delivering a normal living calf. During late pregnancy and at the time of parturition, the levels of E1SO4 and PAGs in animals delivering a stillborn or weak calf (from group A1) followed the normal profiles found in animals delivering a normal living calf. In group A2, low levels of E1SO4 and pregnancy associated glycoproteins (PAGs) over 24 h at both 3 and 6 weeks prior to parturition (<1.5 nmol/L) were recorded in animals delivering a stillborn calf. During late pregnancy and parturition, the levels of E1SO4 and PAGs were slightly lower during 30–50 days prior to delivery and increased with a lower magnitude at the time of parturition. In conclusion, our results indicate that the aetiology behind stillbirth varies depending on the AI-bulls used and is associated with dystocia or low viability of the calves. Deviating profiles of oestrone sulphate (E1SO4) and pregnancy associated glycoproteins (PAGs) in animals delivering a stillborn calf not caused by dystocia were observed, suggesting placental dysfunction as a possible factor. The finding suggests that the analyses of E1SO4 and PAGs could be used for monitoring foetal well-being in animals with a high risk of stillbirth at term.     

NMR hydrogen exchange of the OB-fold protein LysN as a function of denaturant: the most conserved elements of structure are the most stable to unfolding.

The structure of LysN contains an OB-fold motif composed of a structurally conserved five-stranded beta-barrel capped by a poorly conserved alpha-helix between strands beta3 and beta4. Two additional alpha-helices, unique to the LysN structure, flank the N terminus of the OB-fold. The stability of LysN to unfolding has been investigated with NMR native state hydrogen exchange measurements as a function of guanidinium hydrochloride concentration, and equilibrium unfolding transitions monitored by ellipticity at 222 nm and fluorescence at 350 nm. The spectrophotometric measurements suggest an apparent two-state unfolding transition with DeltaGu(0) approximately 6 kcal/mol and m approximately 3 kcal/(molM). By contrast, NMR hydrogen exchange measurements manifest a distribution of DeltaGu(0) and m values which indicate that the protein can undergo subglobal unfolding. The largest DeltaGu(0) values from hydrogen exchange are for residues in the beta-sheet of the protein. These values, which reflect complete unfolding of the protein, are between 3 and 4 kcal/mol higher than those obtained from circular dichroism or fluorescence. This discrepancy may be due to the comparison of NMR hydrogen exchange parameters measured at residue-level resolution, with spectrophotometric parameters that reflect an unresolved super position of unfolding transitions of the alpha-helices and beta-strands. The largest DeltaGu(0) values obtained from hydrogen exchange for the subset of residues in the alpha-helices of the protein, agree with the DeltaGu(0) values obtained from circular dichroism or fluorescence. Based on the hydrogen exchange data, however, the three alpha-helices of LysN are on average 3 kcal/mol less stable than the beta-sheet. Consistent with the subglobal unfolding of LysN evinced by hydrogen exchange, a deletion mutant that lacks the first alpha-helix of the protein retains a cooperatively folded structure. Taken together with previous results on the OB-fold proteins SN and CspA, the present results for LysN suggest that the most conserved elements of structure in the OB-fold motif are the most resistant to denaturation. In all three proteins, stability to denaturation correlates with sequence hydrophobicity.     

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

Investigation of native isolates of entomopathogenic fungi for the biological control of three citrus pests

The pathogenicity of 15 isolates of Beauveria bassiana (Balsamo) Vuillemin, five isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and one isolate of M. flavoviride (Gams and Rozsypal) were tested under laboratory conditions against the subterranean life stages of the citrus pests, Ceratitis rosa Karsch, C. capitata Wiedemann (Diptera: Tephritidae) and Thaumatotibia leucotreta Meyrick. (Lepidoptera: Tortricidae). When these citrus pests were treated with a concentration of 1×10⁷ conidia mL^?1, fungal isolates had a significantly greater effect on the adults of C. rosa and C. capitata than they did on the puparia of these two fruit fly species. Further, C. rosa and C. capitata did not differ significantly in their response to entomopathogenic fungi when adult and pupal mycosis were considered. Depending on fungal isolate, the percentage of T. leucotreta adults which emerged from fungal treated sand ranged from 5 to 60% and the percentage of pupae with visible signs of mycosis ranged from 21 to 93%. The relative virulence of the four most promising fungal isolates, as well as the commercially available B. bassiana product, BroadBand® (Biological Control Products, South Africa), were compared against one another as log-probit regressions of mortality against T. leucotreta which exhibited a dose-dependent response. The estimated LC₅₀ values of the three most virulent B. bassiana isolates ranged from 6.8×10⁵ to 2.1×10⁶ conidia mL^?1, while those of the least pathogenic ranged from 1.6×10⁷ to 3.7×10⁷ conidia mL^?1.     

Specific localization of scallop gill epithelial calmodulin in cilia

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.     

Changes in genomic methylation patterns during the formation of triploid asexual dandelion lineages

DNA methylation is an epigenetic mechanism that has the potential to affect plant phenotypes and that is responsive to environmental and genomic stresses such as hybridization and polyploidization. We explored de novo methylation variation that arises during the formation of triploid asexual dandelions from diploid sexual mother plants using methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. In dandelions, triploid apomictic asexuals are produced from diploid sexual mothers that are fertilized by polyploid pollen donors. We asked whether the ploidy level change that accompanies the formation of new asexual lineages triggers methylation changes that contribute to heritable epigenetic variation within novel asexual lineages. Comparison of MS‐AFLP and AFLP fragment inheritance in a diploid × triploid cross revealed de novo methylation variation between triploid F₁ individuals. Genetically identical offspring of asexual F₁ plants showed modest levels of methylation variation, comparable to background levels as observed among sibs in a long‐established asexual lineage. Thus, the cross between ploidy levels triggered de novo methylation variation between asexual lineages, whereas it did not seem to contribute directly to variation within new asexual lineages. The observed background level of methylation variation suggests that considerable autonomous methylation variation could build up within asexual lineages under natural conditions.     

Evidence for para Dechlorination of Polychlorobiphenyls by Methanogenic Bacteria

When microorganisms eluted from upper Hudson River sediment were cultured without any substrate except polychlorobiphenyl (PCB)-free Hudson River sediment, methane formation was the terminal step of the anaerobic food chain. In sediments containing Aroclor 1242, addition of eubacterium-inhibiting antibiotics, which should have directly inhibited fermentative bacteria and thereby should have indirectly inhibited methanogens, resulted in no dechlorination activity or methane production. However, when substrates for methanogenic bacteria were provided along with the antibiotics (to free the methanogens from dependence on eubacteria), concomitant methane production and dechlorination of PCBs were observed. The dechlorination of Aroclor 1242 was from the para positions, a pattern distinctly different from, and more limited than, the pattern observed with untreated or pasteurized inocula. Both methane production and dechlorination in cultures amended with antibiotics plus methanogenic substrates were inhibited by 2-bromoethanesulfonic acid. These results suggest that the methanogenic bacteria are among the physiological groups capable of anaerobic dechlorination of PCBs, but that the dechlorination observed with methanogenic bacteria is less extensive than the dechlorination observed with more complex anaerobic consortia.