Prediction of the structure of the complex between the 30S ribosomal subunit and colicin E3 via weighted-geometric docking

Colicin E3 kills Escherichia coli cells by ribonucleolytic cleavage in the 16S rRNA. The cleavage occurs at the ribosomal decoding A-site between nucleotides A1493 and G1494. The breaking of this single phosphodiester bond results in a complete termination of protein biosynthesis leading to cell death. A model structure of the complex of the ribosomal subunit 30S and colicin E3 was constructed by means of a new weighted-geometric docking algorithm, in which interactions involving specified parts of the molecular surface can be up-weighted, allowing incorporation of experimental data in the docking search. Our model, together with available experimental data, predicts the role of the catalytic residues of colicin E3. In addition, it suggests that bound acidic immunity protein inhibits the enzymatic activity of colicin E3 by electrostatic repulsion of the negatively charged substrate.     

N,N′-bis-(2,3-methylenedioxyphenyl)urea and N,N′-bis-(3,4-methylenedioxyphenyl)urea interact with auxin in enhancing root formation of M26 apple (Malus pumila Mill.) stem slices

Stem slices cut from micropropagated cuttings of apple rootstock M26 were cultured in the presence of indole-3-butyric acid (IBA) plus N,N-bis-(2,3-methylenedioxyphenyl)urea or N,N-bis-(3,4-methylenedioxyphenyl)urea, to verify if there was an interaction between them in enhancing root formation. The N,N-bis-(methylenedioxyphenyl)ureas were supplemented after, before and in the simultaneous presence of auxin. Our data demonstrate that only the simultaneous presence of auxin and N,N-bis-(methylenedioxyphenyl)ureas in the culture medium enhanced root formation on M26 stem slices. The percentage of rooted slices obtained in the presence of the mixtures was significantly different from that obtained in the presence of low auxin concentration alone (1µM). Moreover both the percentage of rooted slices and the number of roots per slice obtained in these culture conditions was not significantly different to that of the optimal auxinic treatment in which the auxin concentration was threefold higher.     

An unusual halotolerant alpha-type carbonic anhydrase from the alga Dunaliella salina functionally expressed in Escherichia coli

A 60-kDa, salt-inducible, internally duplicated alpha-type carbonic anhydrase (Dca) is associated with the plasma membrane of the extremely salt-tolerant, unicellular, green alga Dunaliella salina. Unlike other carbonic anhydrases, Dca remains active over a very broad range of salinities (0-4M NaCl), thus representing a novel type of extremely halotolerant enzyme. To elucidate the structural principles of halotolerance, structure-function investigations of Dca have been initiated. Such studies require considerable amounts of the enzyme, and hence, large-scale algal cultivation. Furthermore, the purified enzyme is often contaminated with other, co-purifying algal carbonic anhydrases. Expression in heterologous systems offers a means to produce, and subsequently purify, sufficiently large amounts of Dca required for activity and structural studies. Attempts to over-express Dca in the Escherichia coli BL21(DE3)pLysS strain, after optimizing various expression parameters, produced soluble, but weakly active protein, composed of fully reduced and variably -S-S- cross-linked chains (each of the Dca repeats contains a pair of cysteine residues, presumably forming a disulfide bond). However, when the E. coli Origami B(DE3)pLysS strain was used as a host, a functionally active enzyme with proper disulfide bonds was formed in good yield. Affinity-purified recombinant Dca resembled the native enzyme from D. salina in activity and salt tolerance. Hence, this expression system offers a means of pursuing detailed studies of this extraordinary protein using biochemical, biophysical, and crystallographic approaches.     

New achievements in human cell toxicology: the 20th annual workshop on in vitro toxicology

The 20th Annual Workshop on In Vitro Toxicology (Oxford, UK, September 22-24, 2002) was convened as part of a European meeting entitled Human Cell Culture 2002. The meeting was arranged by the Scandinavian Society for Cell Toxicology (SSCT), the European Tissue Culture Society and the British Prostate Group. Two sessions, which are summarised in this report, were devoted to in vitro toxicology: Human Cell Toxicology and The SSCT Free Paper Session. Outstanding experts in the field of toxicology outlined contemporary approaches in toxicity testing in their lectures. Short oral presentations demonstrated a variety of in vitro model systems and methodologies, which can be useful for investigating human toxicity, as well as for studies on mechanisms of toxicity.     

Light-harvesting complex II pigments and proteins in association with Cbr, a homolog of higher-plant early light-inducible proteins in the unicellular green alga Dunaliella

 Like higher plants, unicellular green algae of the genus Dunaliella respond to light stress by enhanced de-epoxidation of violaxanthin and accumulation of Cbr, a protein homologous to early light-inducible proteins (Elips) in plants. Earlier studies indicated that Cbr was associated with the light-harvesting complex of photosystem II (LHCII) and suggested it acted as a zeaxanthin-binding protein and fulfilled a photo-protective function (Levy et al. 1993, J. Biol. Chem. 268: 20892–20896). To characterize the protein-pigment subcomplexes containing Cbr in greater detail than attained so far, thylakoid membranes from Dunaliella salina grown in high light or normal light were solubilized with dodecyl maltoside and fractionated by isoelectric-focusing. Analysis of the resolved LHCII subcomplexes indicated preferred associations among the four LHCIIb polypeptides and between them and Cbr: subcomplexes including Cbr contained one or two of the more acidic of the four LHCIIb polypeptides as well as large amounts of lutein and zeaxanthin relative to chlorophyll a/b. After sucrose gradient centrifugation, Cbr free of LHCIIb polypeptides was detected together with released pigments; this Cbr possibly originated in subcomplexes dissociated in the course of the analysis. These results agree with the conclusion that Cbr is part of the network of LHCIIb protein-pigment complexes and suggest that the role played by Cbr involves the organization and/or stabilization of assemblies highly enriched in zeaxanthin and lutein. Such assemblies may function to protect PSII from photodamage due to overexcitation. Received: 6 August 1999 / Accepted: 23 November 1999     

How to deal with ground truthing affected by human‐induced habitat change?: Identifying high‐quality habitats for the Critically Endangered Red Siskin

Species distribution models (SDM) can be valuable for identifying key habitats for conservation management of threatened taxa, but anthropogenic habitat change can undermine SDM accuracy. We used data for the Red Siskin (Spinus cucullatus), a critically endangered bird and ground truthing to examine anthropogenic habitat change as a source of SDM inaccuracy. We aimed to estimate: (1) the Red Siskin's historic distribution in Venezuela; (2) the portion of this historic distribution lost to vegetation degradation; and (3) the location of key habitats or areas with both, a high probability of historic occurrence and a low probability of vegetation degradation. We ground‐truthed 191 locations and used expert opinion as well as landscape characteristics to classify species' habitat suitability as excellent, good, acceptable, or poor. We fit a Random Forest model (RF) and Enhanced Vegetation Index (EVI) time series to evaluate the accuracy and precision of the expert categorization of habitat suitability. We estimated the probability of historic occurrence by fitting a MaxLike model using 88 presence records (1960–2013) and data on forest cover and aridity index. Of the entire study area, 23% (20,696 km²) had a historic probability of Red Siskin occurrence over 0.743. Furthermore, 85% of ground‐truthed locations had substantial reductions in mean EVI, resulting in key habitats totaling just 976 km², in small blocks in the western and central regions. Decline in Area of Occupancy over 15 years was between 40% and 95%, corresponding to an extinction risk category between Vulnerable and Critically Endangered. Relating key habitats with other landscape features revealed significant risks and opportunities for proposed conservation interventions, including the fact that ongoing vegetation degradation could limit the establishment of reintroduced populations in eastern areas, while the conservation of remaining key habitats on private lands could be improved with biodiversity‐friendly agri‐ and silviculture programs.     

Splenocytes Seed Bone Marrow of Myeloablated Mice: Implication for Atherosclerosis

Extramedullary hematopoiesis has been shown to contribute to the pathogenesis of a variety of diseases including cardiovascular diseases. In this process, the spleen is seeded with mobilized bone marrow cells that augment its hematopoietic ability. It is unclear whether these immigrant cells that are produced/reprogrammed in spleen are similar or different from those found in the bone marrow. To begin to understand this, we investigated the relative potency of adult splenocytes per se to repopulate bone marrow of lethally-irradiated mice and its functional consequences in atherosclerosis. The splenocytes were harvested from GFP donor mice and transplanted into myeloablated wild type recipient mice without the inclusion of any bone marrow helper cells. We found that adult splenocytes repopulated bone marrow of myeloablated mice and the transplanted cells differentiated into a full repertoire of myeloid cell lineages. The level of monocytes/macrophages in the bone marrow of recipient mice was dependent on the cell origin, i.e., the donor splenocytes gave rise to significantly more monocytes/macrophages than the donor bone marrow cells. This occurred despite a significantly lower number of hematopoietic stem cells being present in the donor splenocytes when compared with donor bone marrow cells. Atherosclerosis studies revealed that donor splenocytes displayed a similar level of atherogenic and atheroprotective activities to those of donor bone marrow cells. Cell culture studies showed that the phenotype of macrophages derived from spleen is different from those of bone marrow. Together, these results demonstrate that splenocytes can seed bone marrow of myeloablated mice and modulate atherosclerosis. In addition, our study shows the potential of splenocytes for therapeutic interventions in inflammatory disease.     

Active anthocyanin degradation in <Emphasis Type="Italic">Brunfelsia calycina</Emphasis> (yesterday–today–tomorrow) flowers

Anthocyanins are the largest group of plant pigments responsible for colors ranging from red to violet and blue. The biosynthesis of anthocyanins, as part of the larger phenylpropanoid pathway, has been characterized in great detail. In contrast to the detailed molecular knowledge available on anthocyanin synthesis, very little is known about the stability and catabolism of anthocyanins in plants. In this study we present a preliminary characterization of active in planta degradation of anthocyanins, requiring novel mRNA and protein synthesis, in Brunfelsia calycina flowers. Brunfelsia is a unique system for this study, since the decrease in pigment concentration in its flowers (from dark purple to white) is extreme and rapid, and occurs at a specific and well-defined stage of flower development. Treatment of detached flowers with protein and mRNA synthesis inhibitors, at specific stages of flower development, prevented degradation. In addition, treatment of detached flowers with cytokinins delayed senescence without changing the rate of anthocyanin degradation, suggesting that degradation of anthocyanins is not part of the general senescence process of the flowers but rather a distinctive and specific pathway. Based on studies on anthocyanin degradation in wine and juices, peroxidases are reasonable candidates for the in vivo degradation. A significant increase in peroxidase activity was shown to correlate in time with the rate of anthocyanin degradation. An additional indication that oxidative enzymes are involved in the process is the fact that treatment of flowers with reducing agents, such as DTT and glutathione, caused inhibition of degradation. This study represents the first step in the elucidation of the molecular mechanism behind in vivo anthocyanin degradation in plants.     

Genetic Diversity and Spatial Genetic Structure of Chamaedaphne calyculata (Ericaceae) at the Western Periphery in Relation to its Main Continuous Range in Eurasia

A previous phylogeography and genetic diversity study of Chamaedaphne calyculata (Ericaceae) showed that populations over its geographic range were strongly separated into two groups: a Eurasian/NW North American group and a NE North American one corresponding with the disjunct distribution of Sphagnum-dominated peatlands in north-western and central-eastern North America. Here, I have extended the survey and focused on the species’ detailed postglacial origin and the effect of isolation on genetic diversity patterns, particularly within island-like populations at the western periphery of its range in Europe. Using AFLP markers, estimates of genetic diversity within 16 C. calyculata populations in the Eurasian group were low (percentage of polymorphic loci P _PL=14.9–24.8 %, Nei’s gene diversity H=0.060–0.119). Genetic diversity patterns within this species did not support the hypothesis that genetic diversity decreases towards the periphery of the range. Bayesian clustering analysis showed that population-level admixture was present in almost all studied 16 populations, suggesting multi-directional gene flow. On the other hand, the majority of assigned individuals (ca. 98 % of individuals) were offspring of the original residents, confirming that C. calyculata populations in the present day acted as discrete genetic units both in its continuous range and at its western periphery, and that gene flow was historic rather than contemporary in Eurasia. There was no correlation between genetic and geographic distance in the Eurasian group (r=0.02, P>0.05, Mantel test) nor at the western periphery (r=0.15, P>0.05, Mantel test). The isolation-by-distance (IBD) scatterplot matched Hutchinson and Templeton’s interpretation (case III), and geographic distance between populations was not a reliable predictor of the degree of genetic differentiation between populations. It is suggested that the lack of IBD might be a result of random genetic drift in rather disconnected populations that have become increasingly fragmented relatively recently. Positive and significant relationships between genetic and geographic distance on a small population scale was the result of biparental inbreeding of C. calyculata and restricted seed rain. Despite sporadic generative reproduction and limited dispersal, the fine-scale genetic structure within populations has been maintained, even though population sizes have been reduced to small fragments in recent years.