Non-histone protein methylation in Trypanosoma cruzi epimastigotes

Post-translational methylation of proteins, which occurs in arginines and lysines, modulates several biological processes at different levels of cell signaling. Recently, methylation has been demonstrated in the regulation beyond histones, for example, in the dynamics of protein-protein and protein-nucleic acid interactions. However, the presence and role of non-histone methylation in Trypanosoma cruzi, the etiologic agent of Chagas disease, has not yet been elucidated. Here, we applied mass spectrometry-based-proteomics (LC-MS/MS) to profile the methylproteome of T. cruzi epimastigotes, describing a total of 1252 methyl sites in 824 proteins. Functional enrichment and protein-protein interaction analysis show that protein methylation impacts important biological processes of the parasite, such as translation, RNA and DNA binding, amino acid, and carbohydrate metabolism. In addition, 171 of the methylated proteins were previously reported to bear phosphorylation sites in T. cruzi, including flagellar proteins and RNA binding proteins, indicating that there may be an interplay between these different modifications in non-histone proteins. Our results show that a broad spectrum of functions is affected by methylation in T. cruzi, indicating its potential to impact important processes in the biology of the parasite and other trypanosomes.     

A vegetation map of tropical continental Asia at scale 1:5 million

Abstract. A vegetation map at scale 1:5 million is presented. * * Attached on the inside of this issue's back cover.
It covers Bangladesh, Burma (Myanmar), India, Cambodia, Laos, Thailand, Vietnam and Sri Lanka and fills a conspicuous gap in the cartography of tropical vegetation, following the publication of vegetation maps of South America, Africa and Malaysia. For conformity, it is presented as one sheet at a scale of 1:5 million. It uses the basic map of the American Geographical Society (1942; bipolar oblique conformal projection) which forms the base for FAO's Soil map of the world. Basic information was obtained from many published maps, unpublished observations and satellite data. The limits of the main vegetation types have been updated with a complete set of Landsat MSS images (369 scenes) with a mosaic of Landsat TM data for 1991 and with recent forest maps from Asia. Nine main vegetation units, which are groups of forest formations, have been identified and mapped, including woodlands, thickets and wooded savannas. Agricultural land has been shown in a uniform pale green colour in order to clearly express the extent of human impacts on woody vegetation. In spite of the necessary oversimplification of the ground data, this map is probably the most explicit expression of the remaining forest stands and of the regression of natural vegetation in the region. It can be considered as a benchmark for future monitoring of tropical vegetation.     

Activity of cell wall-associated enzymes in ripening olive fruit

Enzymatically active cell wall isolaled from olive (Olea europaea) fruit was employed Hi investigate some hydrolytic enzymes bound to the cell wall and the changes in these during ripening. Seven glycosidases. β-glucosidase (EC 3.2.1.21) α-galactosidase (EC 3.2.1.22). β-galactosidase (EC 3.2.1.23). α-arabinosidase (EC 3.2.1.55), α-mannosidase (EC 3.2.1,24). β-xylosidase (EC 3.2.1.37) and β-N-acetylglucosamidase (EC 3.2.1.30). as well as Cx-cellulase (EC 3.2.1.4) and endo-polygalacturonase (EC 3.2.1.15). were identified in the cell wall preparation, at four stages of ripeness (mature green. changing colour, black and black-ripe). Activities of all these cell wall-associated enzymes fionicallv and covalently linked) were determined either by cell wall incubation with artificial substrate or after extraction from the cell wall with buffers of high salt concentration (Cx-cellulase). and were compared to those of forms solubilized from acetone powders with 500 nM citrate buffer (cytoplasmic and/or apoplastic plus ionically hound to cell wall) In general, the activities of low ionic strength buffer-soluble enzymes were found to be much higher than those of the bound enzymes. The bound enzymes are present in the fruit at the green colour stage, whereas the activities of the soluble enzymes only increased from the changing colour stage onwards. The tenacity of binding of enzymes to the wall was investigated by treating the walls with high salt and measuring residual activity. The nature of the ionic and covalent binding and the changes during ripening were also established for wall-hound glycosidase During ripening there was a marked change in the percentages of covalently- and tonically linked activities of β-glucosidase and β-galaclosidase: al the changing colour stages about 75–80% of the bound active in was present in high ionic strength buffer while al the black-ripe stage it was only 15–20. A possible role for these cell wall degradative enzymes in olive softening is discussed.     

Assessment of various commercial enzymes in the bleaching of radiata pine kraft pulps

Four xylanase preparations that are commercially available, namely Cartazyme from Sandoz, Ecopulp from Alko-ICI, Irgazyme from Ciba-Genencor and Pulpzyme HB from Novo Nordisk, were tested in bleaching experiments of kraft pulps from Pinus radiata. The main objective of this study was to optimize a reduction in the consumption of chlorine dioxide in the bleaching sequences C₉₀/D₁₀E_oDED, C₇₀/D₃₀E_oDED and D₁₀₀EDED. Enzymatic treatments led to savings of ClO₂ between 3.5 and 3.9 kg per air-dried tons (ADT) in the three bleaching sequences, without affecting the target brightness of the pulps. In these assays, some minor although reproducible differences in the performance of the enzymes were observed. In most cases, xylanase treatment partially affected the beatability of the pulps, measured as the number of revolutions in the PFI mill required to reach the same tensile index as the respective controls.     

TCEN-49, a monoclonal antibody that identifies a central body antigen in the planarian Dugesia (Girardia) tigrina. Implications for pattern formation and positional signalling mechanisms

We have produced monoclonal antibodies (mAbs) against antigens of the freshwater planarian Dugesia (G.) tigrina (Girard) using standard protocols. One of these mAbs, TCEN-49, detects an antigen (TCEN-49Ag) present in most cells of the central area of the body, including the pharynx. Labelled cells seem more related by position than by lineage, suggesting that TCEN-49Ag is involved somehow in the expression of central body positional identity. The spatial and temporal changes in TCEN-49Ag expression during growth/degrowth and regeneration have been monitored and the implications of these results are discussed.     

The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout,Oncorhynchus mykiss

The complete nucleotide sequence of the mitochondrial DNA of the rainbow trout, Onchorynchus mykiss, has been determined. The total length of the molecule is 16,660 bp. The rainbow trout mitochondrial DNA has the same organization described in eutherian mammals, the clawed frog (Xenopus laevis), and the two fish species, Oriental stream loach (Crossotoma lacustre) and carp (Cyprinus carpio). Alignment and comparison of the deduced amino acid sequences of the 13 proteins encoded by rainbow trout and other vertebrate mitochondrial genomes allowed us to estimate that COI is the most conserved mitochondrial subunit (amino acid identity ranging from 85.6% to 94.8%) whereas ATPase 8 is the most variable one (amino acid identity ranging from 30.8% to 70.4%). Putative secondary structures for the 22 tRNAs found in the molecule are given along with an extensive comparison of tRNA sequences among representative species of each major group of vertebrates. In this sense, an unusual cloverleaf structure for the tRNA^Ser(AGY) is proposed. A stem-loop structure inferred for the origin of the L-strand replication (O_L) and the presence of a large polycytidine tract in the O_L loop is described. The existence of this stretch instead of the usual T-rich sequence reported so far in mammal mtDNAs is explained in terms of a less-strict template dependence of the RNA primase involved in the initiation of L-strand replication. Correspondence to: J.M. Bautista     

Peroxynitrite-Induced Cytotoxicity in PC12 Cells: Evidence for an Apoptotic Mechanism Differentially Modulated by Neurotrophic Factors

Abstract: Peroxynitrite is a powerful oxidant formed by the near-diffusion-limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (>2 m M ) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC₅₀ = 850 µ M ) initially (3–4 h) caused minimal damage to low-density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite-treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite-treated cells demonstrated DNA fragmentation (by terminal-deoxynucleotide transferase-mediated dUTP-digoxigenin nick end-labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite-induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite-initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite-induced apoptosis in vitro.     

Regulation of L-Type Calcium Channels in GH4 Cells via A1 Adenosine Receptors

Abstract: Identification of A₁ adenosine receptors (A₁Rs) in a tumor cell line derived from rat pituitary (GH₄ cells) was performed by ligand binding and immunological experiments. Subsequently, the involvement of A₁Rs in the regulation of calcium conductance was studied in these cells. The agonist N ⁶-( R )-(2-phenylisopropyl)adenosine ( R -PIA) did not modify the intracellular calcium basal levels, whereas it inhibited the increase produced by 15 m M KCl depolarization. The antagonist 1,3-dipropyl-8-cyclopentylxanthine led to the opening of voltage-dependent cell surface calcium channels in the absence of exogenous KCl. The channels were of the L type because the effect was abolished by calciseptine and by verapamil. These results suggest that endogenous adenosine exerts a tonic inhibitory effect on calcium transport. This was confirmed by the high adenosine concentration found in cell supernatants (up to 1 µ M ) and by the calcium mobilization produced by exogenously added adenosine deaminase. In depolarizing conditions, the calcium peak in the presence of adenosine deaminase was reduced when cells were preincubated with R -PIA, thus suggesting that A₁R activation regulates the intensity of depolarization. These results demonstrate that adenosine is an important regulator of the physiological state of pituitary tumor cells by modulating, in an autocrine manner, the activity of L-type voltage-dependent calcium channels.     

Biodiversity among West African Rhizophora: Foliar wax chemistry

Foliage from 21 red mangroves (Rhizophora mangle, R. racemosa and R. x harrisonii) from different ecogeographic conditions in Gabon, West Africa, was analyzed for epicuticular wax composition using CC, GC and GC-MS. Aliphatic hydrocarbons ranging from C₂₃ to C₃₄ and some triterpenoids were identified. Alkanes were the major constituents (46.7–99.9%), with triterpenoids also accounting for up to 53.3% of the wax extract. High contents of octacosane (27.2%) and lower amounts of nonacosane (14.9%) and hentriacontane (9.8%) distinguished R. mangle from the other two species. R. hizophora x harrisonii was exceptionally rich in nonacosane (45.3%) with moderately high concentrations of hentriacontane (25.4%), whereas R. racemosa was intermediate between the two former species in its content of these three alkanes. Inclusion of the less abundant constituents in principal components analysis provided a good separation of R. x harrisonii, whereas the two other species showed some degree of overlap. Although major alkane patterns can be used to discriminate among the Rhizophora species examined, we do find substantial intra-specific variation that may be attributable to population genetic variation; this was least for R. x harrisonii. The hybrid status of this latter species could not be confirmed from the biochemical analyses carried out.