Novobiocin-Induced Ultrastructural Changes and Antagonism of DNA Synthesis in Trypanosoma cruzi Amastigotes Growing in Cell-Free Medium

The antimicrobial agent novobiocin, an inhibitor of the bacterial enzyme topoisomerase II (DNA gyrase), is known to antagonize Trypanosoma cruzi amastigotes growing in cell-free medium. To determine sites of antagonism of novobiocin, the effects of drug on parasite ultrastructure and incorporation of radiolabeled precursors of DNA, RNA and protein into macromolecules were determined. The predominant ultrastructural abnormality seen after exposure to 0.40 mM novobiocin for 24 h was the presence of electron-dense clumps in the mitochondrion-kinetoplast organelle in 95 of 257 (37%) of cells, in comparison to no clumps seen in 110 drug-free cells. In addition, in the nucleus, the karyosome was less distinct than in control cells and appeared to merge with the chromatin. In the radiolabcling studies, incorporation of thymidine was inhibited in a dose-dependent fashion by novobiocin (0.16–0.80 mM) in a range of drug concentrations that also inhibited parasite growth. For 0.16 and 0.24 mM novobiocin, incorporation of thymidine was inhibited up to 65% relative to drug-free control cells while uptake of uridine and leucine was unaltered. We interpret these ultrastructure and precursor-incorporation studies as suggesting that (i) the mitochondrion-kinetoplast and possibly the nucleus are sites of novobiocin antagonism of T. cruzi amastigotes and (ii) that novobiocin appears to antagonize DNA synthesis within these organisms. Whether the drug target is topoisomerase II, however, is as yet unknown.     

The 44-kDa Regulatory Subunit of the Paramecium cAMP-Dependent Protein Kinase Lacks a Dimerization Domain and May Have a Unique Autophosphorylation Site Sequence

ABSTRACT. The 44-kDa regulatory subunit (R₄₄) of one form of cAMP-dependent protein kinase of Paramecium was purified, and two partial internal amino acid sequences from it were used to clone the corresponding cDNA. This R₄₄ cDNA clone was 1022-bp long, including 978 bp of coding sequence and 7 bp and 37 bp of 5' and 3' untranslated sequences, respectively. A 1.1-kb mRNA was labeled on a Northern blot. The deduced R₄₄ amino acid sequence had 31%–38% positional identity to the sequences of other cloned cAMP-dependent protein kinase regulatory subunits. R₄₄ sequence showed equal sequence similarity to mammalian types I and II regulatory subunits. The N -terminal sequence encoding the regulatory subunit dimerization domain found in most regulatory subunits is not present in the R₄₄ clone, confirming the lack of regulatory subunit dimer formation previously reported for the Paramecium cAMP-dependent protein kinase. The putative autophosphorylation site of R₄₄ contains the amino acid sequence TRTS, distinct from the consensus sequence RRXS, where X is any residue, found in other autophosphorylated cAMP-dependent protein kinase regulatory subunits and many cAMP-dependent protein kinase substrates.     

Contrasting effects of tropospheric ozone on five native herbs which coexist in calcareous grassland

The aim of this study was to examine the effects of increased tropospheric ozone concentrations on the growth and morphology of five native herbs commonly found to coexist in calcareous grassland in areas of Britain and continental Europe: Anthyllis vulneraria L., Cirsium acaule (L.) Scop., Festuca ovina L., Pilosella offtcinarum F. Shultz & Shultz-Bip and Lotus comiculatus L. In a chronic fumigation (mean O₃ concentration of 71 ppb (71 nl 1^?1) for 7 h d^?1 AOT40 4585 ppb-h) which lasted for 21 d, the effects of ozone were assessed using classical growth analysis. Large reductions in mean relative growth rates for shoot and root weight and root length were observed for the two legumes (Fabaceae) Lotus corniculatus and Anthyllis vulneraria, although these were only statistically significant for Lotus corniculatus. Significant reductions in specific root length (length per unit dry weight) were found for Cirsium acaule and Pilosella officinarum (Asteraceae), while for Festuca ovina (Poaceae) the allometric coefficient was reduced significantly following exposure to ozone. An acute fumigation (mean O₃ concentration of 196 ppb, 7 h) resulted in a range of visible injury, from no injury (Festuca ovina and Pilosella officinarum) through moderate levels of injury (Cirsium acaule and Lotus corniculatus) to extensive and widespread injury (Anthyllis vulneraria). Scoring of visible damage showed that this was only statistically significant for the two legumes, Lotus corniculatus and Anthyllis vulneraria. These results suggest that native herbs may differ in their sensitivity to tropospheric ozone. Both chronic and acute exposures revealed that members of the Fabaceae may be most sensitive to ozone pollution, but the study also suggests that subtle changes in root morphology occurred for members of the Asteraceae. These findings are discussed in relation to the critical levels of ozone set recently for plants and the implications of increasing tropospheric ozone for the conservation of native plant communities.     

A Mutation in Vicia faba Results in Ineffective Nodules with Impaired Bacteroid Differentiation and Reduced Synthesis of Late Nodulins

Although numerous reports have documented the effect of bacterially-inducedineffectiveness on root nodule structure, function, and plantgene expression, few studies have detailed the effect of theplant genome on similar parameters. In this report effective(N₂-fixing) broadbean {Vicia faba L.) and plant-controlled ineffective(non-N₂-fixing) broadbean recessive for the sym-1 gene werecompared for nodule structure, developmental expression of noduleenzyme activities, enzyme proteins, and mRNAs involved in Nassimilation, leghemoglobin (Lb) synthesis, and acetylene reductionactivity (ARA). During development of effective wild-type nodules,glutamine synthetase (GS), aspartate aminotransferase (AAT),phosphoenolpyruvate carboxylase (PEPC) and NADH-glutamate synthase(GOGAT) activities and enzyme proteins increased coincidentwith nodule ARA. The increases in GS, AAT, and PEPC were associatedwith increased synthesis of mRNAs for these proteins. Synthesisof Lb polypeptides and mRNAs during development of effectivenodules was similar to that of GS, AAT, and PEPC. By contrast,ineffective sym-1 nodules displayed little or no ARA and hadneither the increases in enzyme activities nor enzyme proteinsand mRNAs as seen for effective nodules. The effect of the sym-1gene appeared to occur late in nodule development at eitherthe stage of bacterial release from infection threads or differentiationof bacteria into bacteroids. High in vitro enzyme activities,enzyme polypeptides, and mRNA levels in parental effective noduleswere dependent upon a signal associated with effective bacteroidsthat was lacking in sym-1 nodules. Nodule organogenesis didnot appear to be a signal for the induction of GS, PEPC, AAT,and Lb expression in sym-1 nodules. Key words: Vicia faba, mutation, sym-1 gene, nodules     

Paramecium Has Two Regulatory Subunits of Cyclic AMP-Dependent Protein Kinase, One Unique to Cilia

ABSTRACT. The subunit composition and intracellular location of the two forms of cAMP-dependent protein kinase of Paramecium cilia were determined using antibodies against the 40-kDa catalytic (C) and 44-kDa regulatory (R₄₄) subunits of the 70-kDa cAMP-dependent protein kinase purified from deciliated cell bodies. Both C and R₄₄ were present in soluble and particulate fractions of cilia and deciliated cells. Crude cilia and a soluble ciliary extract contained a 48-kDa protein (R₄₈) weakly recognized by one of several monoclonal antibodies against R₄₄, but not recognized by an anti-R₄₄ polyclonal serum. Gel-filtration chromatography of a soluble ciliary extract resolved a 220-kDa form containing C and R₄₈ and a 70-kDa form containing C and R₄₄. In the large enzyme, R₄₈ was the only protein to be autophosphorylated under conditions that allow autophosphorylation of R₄₄ The subunits of the large enzyme subsequently were purified to homogeneity by cAMP-agarose chromatography. Both C and R₄₈ were retained by the column and eluted with 1 M NaCl; no other proteins were purified in this step. These results confirm that the ciliary cAMP-dependent protein kinases have indistinguishable C subunits, but different R subunits. The small ciliary enzyme, like the cell-body enzyme, contains R₄₄, whereas R₄₈ is the R subunit of the large enzyme.     

Technical note

Modifications to a French pressure cell, designed for the disruption of microbes, are described which allow the apparatus to be used for the incubation of anaerobic bacteria at temperatures and pressures up to 75°C and 500 bar.     

Greenhouse gas emissions from four bioenergy crops in England and Wales: Integrating spatial estimates of yield and soil carbon balance in life cycle analyses

Accurate estimation of the greenhouse gas (GHG) mitigation potential of bioenergy crops requires the integration of a significant component of spatially varying information. In particular, crop yield and soil carbon (C) stocks are variables which are generally soil type and climate dependent. Since gaseous emissions from soil C depend on current C stocks, which in turn are related to previous land management it is important to consider both previous and proposed future land use in any C accounting assessment. We have conducted a spatially explicit study for England and Wales, coupling empirical yield maps with the RothC soil C turnover model to simulate soil C dynamics. We estimate soil C changes under proposed planting of four bioenergy crops, Miscanthus ( Miscanthus × giganteus ), short rotation coppice (SRC) poplar ( Populus trichocarpa Torr. & Gray × P. trichocarpa , var. Trichobel), winter wheat, and oilseed rape. This is then related to the former land use – arable, pasture, or forest/seminatural, and the outputs are then assessed in the context of a life cycle analysis (LCA) for each crop. By offsetting emissions from management under the previous land use, and considering fossil fuel C displaced, the GHG balance is estimated for each of the 12 land use change transitions associated with replacing arable, grassland, or forest/seminatural land, with each of the four bioenergy crops. Miscanthus and SRC are likely to have a mostly beneficial impact in reducing GHG emissions, while oilseed rape and winter wheat have either a net GHG cost, or only a marginal benefit. Previous land use is important and can make the difference between the bioenergy crop being beneficial or worse than the existing land use in terms of GHG balance.     

The Regulation of Dictyostelium Development by Transmembrane Signalling

ABSTRACT. Dictyostelium discoideum has a well characterized life cycle where unicellular growth and multicellular development are separated events. Development is dependent upon signal transduction mediated by cell surface, cAMP receptor/G protein linkages. Secreted cAMP acts extracellularly as a primary signal and chemoattractant. There are 4 genes for the distinct cAMP receptor subtypes, CAR1, CAR2, CAR3 and CAR4. These subtypes are expressed with temporally and spatially specific patterns and cells carrying null mutations for each gene have distinct developmental phenotypes. These results indicate an essential role for cAMP signalling throughout Dictyostelium development to regulate such diverse pathways as cell motility, aggregation (multicellularity), cytodifferentiation, pattern formation and cell type-specific gene expression.