Serratia marcescens chitinase: One-step purification and use for the determination of chitin

The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.     

Connective Auxin Transport in the Shoot Facilitates Communication between Shoot Apices

The bulk polar movement of the plant signaling molecule auxin through the stem is a long-recognized but poorly understood phenomenon. Here we show that the highly polar, high conductance polar auxin transport stream (PATS) is only part of a multimodal auxin transport network in the stem. The dynamics of auxin movement through stems are inconsistent with a single polar transport regime and instead suggest widespread low conductance, less polar auxin transport in the stem, which we term connective auxin transport (CAT). The bidirectional movement of auxin between the PATS and the surrounding tissues, mediated by CAT, can explain the complex auxin transport kinetics we observe. We show that the auxin efflux carriers PIN3, PIN4, and PIN7 are major contributors to this auxin transport connectivity and that their activity is important for communication between shoot apices in the regulation of shoot branching. We propose that the PATS provides a long-range, consolidated stream of information throughout the plant, while CAT acts locally, allowing tissues to modulate and be modulated by information in the PATS.     

Engineering tolerance and accumulation of lead and cadmium in transgenic plants

We have studied the utility of the yeast protein YCF1, which detoxifies cadmium by transporting it into vacuoles, for the remediation of lead and cadmium contamination. We found that the yeast YCF1-deletion mutant DTY167 was hypersensitive to Pb(II) as compared with wild-type yeast. DTY167 cells overexpressing YCF1 were more resistant to Pb(II) and Cd(II) than were wild-type cells, and accumulated more lead and cadmium. Analysis of transgenic Arabidopsis thaliana plants overexpressing YCF1 showed that YCF1 is functionally active and that the plants have enhanced tolerance of Pb(II) and Cd(II) and accumulated greater amounts of these metals. These results suggest that transgenic plants expressing YCF1 may be useful for phytoremediation of lead and cadmium.     

Mechanism of the Anticoagulant Activity of Thrombin Mutant W215A/E217A

The thrombin mutant W215A/E217A (WE) is a potent anticoagulant both in vitro and in vivo. Previous x-ray structural studies have shown that WE assumes a partially collapsed conformation that is similar to the inactive E* form, which explains its drastically reduced activity toward substrate. Whether this collapsed conformation is genuine, rather than the result of crystal packing or the mutation introduced in the critical 215–217 β-strand, and whether binding of thrombomodulin to exosite I can allosterically shift the E* form to the active E form to restore activity toward protein C are issues of considerable mechanistic importance to improve the design of an anticoagulant thrombin mutant for therapeutic applications. Here we present four crystal structures of WE in the human and murine forms that confirm the collapsed conformation reported previously under different experimental conditions and crystal packing. We also present structures of human and murine WE bound to exosite I with a fragment of the platelet receptor PAR1, which is unable to shift WE to the E form. These structural findings, along with kinetic and calorimetry data, indicate that WE is strongly stabilized in the E* form and explain why binding of ligands to exosite I has only a modest effect on the E*-E equilibrium for this mutant. The E* → E transition requires the combined binding of thrombomodulin and protein C and restores activity of the mutant WE in the anticoagulant pathway.Thrombin is the pivotal protease of blood coagulation and is endowed with both procoagulant and anticoagulant roles in vivo (1). Thrombin acts as a procoagulant when it converts fibrinogen into an insoluble fibrin clot, activates clotting factors V, VIII, XI, and XIII, and cleaves PAR1² and PAR4 on the surface of human platelets thereby promoting platelet aggregation (2). Upon binding to thrombomodulin, a receptor present on the membrane of endothelial cells, thrombin becomes unable to interact with fibrinogen and PAR1 but increases >1,000-fold its activity toward the zymogen protein C (3). Activated protein C generated from the thrombin-thrombomodulin complex down-regulates both the amplification and progression of the coagulation cascade (3) and acts as a potent cytoprotective agent upon engagement of EPCR and PAR1 (4).The dual nature of thrombin has long motivated interest in dissociating its procoagulant and anticoagulant activities (5–12). Thrombin mutants with anticoagulant activity help rationalize the bleeding phenotypes of several naturally occurring mutations and could eventually provide new tools for pharmacological intervention (13) by exploiting the natural protein C pathway (3, 14, 15). Previous mutagenesis studies have led to the identification of the E217A and E217K mutations that significantly shift thrombin specificity from fibrinogen toward protein C relative to the wild type (10–12). Both constructs were found to display anticoagulant activity in vivo (10, 12). The subsequent discovery of the role of Trp-215 in controlling the balance between pro- and anti-coagulant activities of thrombin (16) made it possible to construct the double mutant W215A/E217A (WE) featuring >19,000-fold reduced activity toward fibrinogen but only 7-fold loss of activity toward protein C (7). These properties make WE the most potent anticoagulant thrombin mutant engineered to date and a prototype for a new class of anticoagulants (13). In vivo studies have revealed an extraordinary potency, efficacy, and safety profile of WE when compared with direct administration of activated protein C or heparin (17–19). Importantly, WE elicits cytoprotective effects (20) and acts as an antithrombotic by antagonizing the platelet receptor GpIb in its interaction with von Willebrand factor (21).What is the molecular mechanism underscoring the remarkable functional properties of WE? The mutant features very low activity toward synthetic and physiological substrates, including protein C. However, in the presence of thrombomodulin, protein C is activated efficiently (7). A possible explanation is that WE assumes an inactive conformation when free but is converted into an active form in the presence of thrombomodulin. The ability of WE to switch from inactive to active forms is consistent with recent kinetic (22) and structural (23, 24) evidence of the significant plasticity of the trypsin fold. The active form of the protease, E, coexists with an inactive form, E*, that is distinct from the zymogen conformation (25). Biological activity of the protease depends on the equilibrium distribution of E* and E, which is obviously different for different proteases depending on their physiological role and environmental conditions (25). The E* form features a collapse of the 215–217 β-strand into the active site and a flip of the peptide bond between residues Glu-192 and Gly-193, which disrupts the oxyanion hole. These changes have been documented crystallographically in thrombin and other trypsin-like proteases such as αI-tryptase (26), the high temperature requirement-like protease (27), complement factor D (28), granzyme K (29), hepatocyte growth factor activator (30), prostate kallikrein (31), prostasin (32, 33), complement factor B (34), and the arterivirus protease nsp4 (35). Hence, the questions that arise about the molecular mechanism of WE function are whether the mutant is indeed stabilized in the inactive E* form and whether it can be converted to the active E form upon thrombomodulin binding.Structural studies of the anticoagulant mutants E217K (36) and WE (37) show a partial collapse of the 215–217 β-strand into the active site that abrogates substrate binding. The collapse is similar to, but less pronounced than, that observed in the structure of the inactive E* form of thrombin where Trp-215 relinquishes its hydrophobic interaction with Phe-227 to engage the catalytic His-57 and residues of the 60-loop after a 10 Å shift in its position (24). These more substantial changes have been observed recently in the structure of the anticoagulant mutant Δ146–149e (38), which has proved that stabilization of E* is indeed a molecular mechanism capable of switching thrombin into an anticoagulant. It would be simple to assume that both E217K and WE, like Δ146–149e, are stabilized in the E* form. However, unlike Δ146–149e, both E217K and WE carry substitutions in the critical 215–217 β-strand that could result into additional functional effects overlapping with or mimicking a perturbation of the E*-E equilibrium. A significant concern is that both structures suffer from crystal packing interactions that may have biased the conformation of side chains and loops near the active site (24). The collapsed structures of E217K and WE may be artifactual unless validated by additional structural studies where crystal packing is substantially different.To address the second question, kinetic measurements of chromogenic substrate hydrolysis by WE in the presence of saturating amounts of thrombomodulin have been carried out (37), but these show only a modest improvement of the k_cat/K_m as opposed to >57,000-fold increase observed when protein C is used as a substrate (7, 37). The modest effect of thrombomodulin on the hydrolysis of chromogenic substrates is practically identical to that seen upon binding of hirugen to exosite I (37) and echoes the results obtained with the wild type (39) and other anticoagulant thrombin mutants (7, 9, 10, 12, 38). That argues against the ability of thrombomodulin alone to significantly shift the E*-E equilibrium in favor of the E form. Binding of a fragment of the platelet receptor PAR1 to exosite I in the D102N mutant stabilized in the E* form (24) does trigger the transition to the E form (23), but evidence that a similar long-range effect exists for the E217K or WE mutants has not been presented.In this study we have addressed the two unresolved questions about the mechanism of action of the anticoagulant thrombin mutant WE. Here we present new structures of the mutant in its human and murine versions, free and bound to a fragment of the thrombin receptor PAR1 at exosite I. The structures are complemented by direct energetic assessment of the binding of ligands to exosite I and its effect on the E*-E equilibrium.     

Root Strength and Root Area Ratio of Forest Species in Lombardy (Northern Italy)

Forest vegetation is known to increase hillslope stability by reinforcing soil shear resistance and by influencing hydrologic conditions of soil. Although the importance of plant root systems for hillslope stability has received considerable attention in recent years, the quantification of such an effect needs more investigation. In this paper, we present a synthesis of the data gathered in the last 5 years for some species in different locations of the Alps and Prealps of Lombardy (Northern Italy) with the aim to increase our knowledge on root tensile strength and on Root Area Ratio distribution within the soil. Concerning root tensile strength we developed tensile strength–diameter relationships for eight species: green alder (Alnus viridis(Chaix) D.C.), beech (Fagus sylvatica L.), red willow (Salix purpurea L.), goat willow (Salix caprea L.), hazel (Corylus avellana L.), European ash (Fraxinus excelsior L.), Norway spruce (Picea abies (L.) Karst.) and European larch (Larix decidua Mill.). Results show a great variability among the different species and also for the same species. In general, however, root strength (in terms of tension) tends to decrease with diameter according to a power law, as observed by other Authors. Comparing the power law fitting curves for the considered species, it can be observed that they fall in a relatively narrow band, with the exception of hazel, which appears the most resistant. Concerning the evaluation of root distribution within the soil we estimated the Root Area Ratio (the ratio between the area occupied by roots in a unit area of soil) according to its depth for five species (beech, Norway spruce, European larch, mixed hazel and ash) in three locations of Lombardy. Results show that there is a great variability of root density for the same species well as for different points at the same locality. The general behaviour of root density, in any case, is to decrease with depth according to a gamma function for all the studied species. The results presented in this paper contribute to expanding the knowledge on root resistance behaviour and on root density distribution within the soil. The studied location have allowed the implementation of soil–root reinforcement models and the evaluation of the vegetation contribution to soil stability.     

Recombinant near-isogenic lines: a resource for the mendelization of heterotic QTL in maize

Although heterosis is widely exploited in agriculture, a clear understanding of its genetic bases is still elusive. This work describes the development of maize recombinant near-isogenic lines (NILs) for the mendelization of six heterotic QTL previously identified based on a maize (Zea mays L.) RIL population. The efficient and inexpensive strategy adopted to generate sets of NILs starting from QTL-specific residual heterozygous lines (RHLs) is described and validated. In particular, we produced nine pairs of recombinant NILs for all six QTL starting from RHLs F_4:5 originally obtained during the production of the RIL population mentioned above. Whenever possible, two different NIL pairs were generated for each QTL. The efficiency of this procedure was tested by analyzing two segregating populations for two of the selected heterotic QTL for plant height, yield per plant and ears per plant. Both additive and dominant effects were observed, consistently with the presence of the QTL within the introgressed regions. Refinement of QTL detection was consistent with previous observations in terms of effects and position of the considered QTL. The genetic material developed in this work represents the starting point for QTL fine mapping aimed at understanding the genetic bases of hybrid vigor in maize. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A room temperature ionic liquid as convenient solvent for the oxidative folding of conopeptides

We report the first example of conopeptide oxidation performed in a biocompatible ionic liquid, 1‐ethyl‐3‐methylimidazolium acetate ([C₂mim][OAc]), which enables the efficient formation of both hydrophilic and poorly water‐soluble conotoxins compared with conventional methods. Moreover, the method features a high‐concentration approach ultimately leading to higher yields at reduced separation effort. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Separate and Combined Effects of DNMT and HDAC Inhibitors in Treating Human Multi-Drug Resistant Osteosarcoma HosDXR150 Cell Line

Understanding the molecular mechanisms underlying multi-drug resistance (MDR) is one of the major challenges in current cancer research. A phenomenon which is common to both intrinsic and acquired resistance, is the aberrant alteration of gene expression in drug-resistant cancers. Although such dysregulation depends on many possible causes, an epigenetic characterization is considered a main driver. Recent studies have suggested a direct role for epigenetic inactivation of genes in determining tumor chemo-sensitivity. We investigated the effects of the inhibition of DNA methyltransferase (DNMT) and hystone deacethylase (HDAC), considered to reverse the epigenetic aberrations and lead to the re-expression of de novo methylated genes in MDR osteosarcoma (OS) cells. Based on our analysis of the HosDXR150 cell line, we found that in order to reduce cell proliferation, co-treatment of MDR OS cells with DNMT (5-Aza-dC, DAC) and HDAC (Trichostatin A, TSA) inhibitors is more effective than relying on each treatment alone. In re-expressing epigenetically silenced genes induced by treatments, a very specific regulation takes place which suggests that methylation and de-acetylation have occurred either separately or simultaneously to determine MDR OS phenotype. In particular, functional relationships have been reported after measuring differential gene expression, indicating that MDR OS cells acquired growth and survival advantage by simultaneous epigenetic inactivation of both multiple p53-independent apoptotic signals and osteoblast differentiation pathways. Furthermore, co-treatment results more efficient in inducing the re-expression of some main pathways according to the computed enrichment, thus emphasizing its potential towards representing an effective therapeutic option for MDR OS.     

Extension of the Messapia x dicoccoides linkage map of Triticum turgidum (L.) Thell

A set of recombinant inbred lines (RIL) derived from a cross between the cultivar Messapia of durum wheat (Triticum turgidum var. durum) and the accession MG4343 of T. turgidum var. dicoccoides was analysed to increase the number of assigned markers and the resolution of the previously constructed genetic linkage map. An updated map of the durum wheat genome consisting of 458 loci was constructed. These loci include 261 Restriction Fragment Length Polymorphisms (RFLPs), 91 microsatellites (Simple Sequence Repeats, SSRs), 87 Amplified Fragment Length Polymorphisms (AFLPs), two ribosomal genes, and nine biochemical (seven seed storage proteins and two isozymes) and eight morphological markers. The loci were mapped on all 14 chromosomes of the A and B genomes, and covered a total distance of 3038.4 cM with an average distance of 6.7 cM between adjacent markers. The molecular markers were evenly distributed between the A and the B genomes (240 and 218 markers, respectively). An additional forty loci (8.8%) could not be assigned to a specific linkage group. A fraction (16.4%) of the markers significantly deviated from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on the 1B, 2A, 2B, 3A, 4A, 7A and 7B chromosomes. The genetic lengths of the chromosomes range from 148.8 cM (chromosome 6B) to 318.0 cM (chromosome 2B) and approximately concur with their physical lengths. Chromosome 2B has the largest number of markers (47), while the chromosomes with the fewest markers are 3A and 6B (23). There are two gaps larger than 40 cM on chromosomes 2A and 3B. The durum wheat map was compared with the published maps of bread and durum wheats; the order of most common RFLP and SSR markers on the 14 chromosomes of the A and B genomes were nearly identical. A core-map can be extracted from the high-density Messapia x dicoccoides map and a subset of uniformly distributed markers can be used to detect and map quantitative trait loci.     

Effects induced by mono- and divalent cations on protein regions responsible for thermal adaptation in β-glycosidase from<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>

The perturbation induced by mono- and divalent cations on the thermophilicity and thermostability of Solfolobus solfataricus -glycosidase, a hyperthermophilic tetrameric enzyme, has been investigated by spectroscopic and computational simulation methods to ascertain the Hofmeister effects on two strategic protein regions identified previously. Specifically, (1) an extra segment (83–124), present only in the sequence of hyperthermophilic glycosidases and recognized as an important thermostability determinant for the enzyme structure; and (2) a restricted area of the subunit interface responsible for the quaternary structure maintenance. Mono- and divalent cations inhibit to a different extent the -glycosidase activity, whose kinetic constants show an apparent competitive inhibition of the catalytic process that reflects the Hofmeister order. The thermostability is also affected by the nature and charge of the cations, reaching maximal effects for the case of Mg²⁺. Fourier transform infrared spectroscopy has revealed very small changes in the protein secondary structure in the presence of the investigated cations at 20 °C, while large effects on the protein melting temperatures are observed. Computational analysis of the enzyme structure has identified negative patches on the accessible surface of the two identified regions. Following the Hofmeister series, cations weaken the existing electrostatic network that links the extra segment to the remaining protein matrix. In particular, the perturbing action of cations could involve the ionic pair interactions E107–R245 and E109–R185, thus leading to a local destructuring of the extra segment as a possible starting event for thermal destabilization. A detailed investigation of the electrostatic network at the A–C intermolecular interface of Sgly after energy minimization suggests that cations could cause a strong attenuation of the ion pair interactions E474–K72 and D473–R402, with consequent partial dissociation of the tetrameric structure.Abbreviations Amide I amide I band in a ²H₂O medium - EM energy minimization - ONPG o-nitrophenyl--d-galactopyranoside - Sgly Escherichia coli expressed Sulfolobus solfataricus -glycosidase