Precise derivatization of structurally distinct chitosans with rhodamine B isothiocyanate

Work to date shows that structurally distinct chitosans have reacted inefficiently and unpredictably with fluorescein isothiocyanate (FITC) in an acid–methanol solvent that maintains both chitosan and fluorophore solubility. Since isothiocyanate preferentially reacts with neutral amine groups, and chitosan solubility typically depends upon a minimal degree of protonation, we tested the hypothesis that precise derivatization of chitosan with rhodamine isothiocyanate (RITC) can be achieved by controlling the reaction time and the degree of protonation. Addition of 50% v/v methanol reduced the chitosan degree of protonation in acetic acid but not HCl solutions. At various degrees of protonation, chitosan reacted inefficiently with RITC as previously observed with FITC. Nevertheless, precise derivatization was achieved by allowing the reaction to proceed overnight at a given degree of protonation (p < 0.0001, n = 18) and fixed initial fluorophore concentration. A reproducible 2% to 4% fraction of neutral amines reacted with RITC in proportion to the initial fluorophore concentration (p < 0.005). Using our optimized protocol, chitosans with different degree of deacetylation and molecular weight were derivatized to either 1% or 0.5% mol/mol RITC/chitosan-monomer with a precision of 0.1% mol/mol. The average molecular weight of fluorescent RITC-chitosan was similar to the unlabeled parent chitosan. Precise molar derivatization of structurally distinct chitosans with RITC can be achieved by controlling chitosan degree of protonation, initial fluorophore concentration, and reaction time.     

Large-scale gene discovery in the septoria tritici blotch fungus Mycosphaerella graminicola with a focus on in planta expression

The foliar disease septoria tritici blotch, caused by the fungus Mycosphaerella graminicola, is currently the most important wheat disease in Europe. Gene expression was examined under highly different conditions, using 10 expressed sequence tag libraries generated from M. graminicola isolate IPO323 using seven in vitro and three in planta growth conditions. To identify fungal clones in the interaction libraries, we developed a selection method based on hybridization with the entire genomic DNA of M. graminicola, to selectively enrich these libraries for fungal genes. Assembly of the 27,007 expressed sequence tags resulted in 9,190 unigenes, representing 5.2 Mb of the estimated 39-Mb genome size of M. graminicola. All libraries contributed significantly to the number of unigenes, especially the in planta libraries representing different stages of pathogenesis, which covered 15% of the library-specific unigenes. Even under presymptomatic conditions (5 days postinoculation), when fungal biomass is less than 5%, this method enabled us to efficiently capture fungal genes expressed during pathogenesis. Many of these genes were uniquely expressed in planta, indicating that in planta gene expression significantly differed from in vitro expression. Examples of gene discovery included a number of cell wall-degrading enzymes, a broad set of genes involved in signal transduction (n=11) and a range of ATP-binding cassette (n=20) and major facilitator superfamily transporter genes (n=12) potentially involved in protection against antifungal compounds or the secretion of pathogenicity factors. In addition, evidence is provided for a mycovirus in M. graminicola that is highly expressed under various stress conditions, in particular, under nitrogen starvation. Our analyses provide a unique window on in vitro and in planta gene expression of M. graminicola.     

Effective production of marker-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene

Public concerns about the issue of the environmental safety of genetically modified plants have led to a demand for technologies allowing the production of transgenic plants without selectable (antibiotic resistance) markers. We describe the development of an effective transformation system for generating such marker-free transgenic plants, without the need for repeated transformation or sexual crossing. This system combines an inducible site-specific recombinase for the precise elimination of undesired, introduced DNA sequences with a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants. The described system can be generally applied to existing transformation protocols, and was tested in strawberry using a model vector in which site-specific recombination leads to a functional combination of a cauliflower mosaic virus 35S promoter and a GUS encoding sequence, thereby enabling the histochemical monitoring of recombination events. Fully marker-free transgenic strawberry plants were obtained following two different selection/regeneration strategies.     

Influence of Dopaminergic Medication on Conditioned Pain Modulation in Parkinson's Disease Patients

Background

Pain is highly prevalent in patients with Parkinson’s disease (PD), but little is known about the underlying pathophysiological mechanisms. The susceptibility to pain is known to depend on ascending and descending pathways. Because parts of the descending pain inhibitory system involve dopaminergic pathways, dysregulations in dopaminergic transmission might contribute to altered pain processing in PD. Deficits in endogenous pain inhibition can be assessed using conditioned pain modulation (CPM) paradigms.

Methods

Applying such a paradigm, we investigated i) whether CPM responses differ between PD patients and healthy controls, ii) whether they are influenced by dopaminergic medication and iii) whether there are effects of disease-specific factors. 25 patients with idiopathic PD and 30 healthy age- and gender-matched controls underwent an established CPM paradigm combining heat pain test stimuli at the forearm and the cold pressor task on the contralateral foot as the conditioning stimulus. PD patients were tested under dopaminergic medication and after at least 12 hours of medication withdrawal.

Results

No significant differences between CPM responses of PD patients and healthy controls or between PD patients “on” and “off” medication were found. These findings suggest (i) that CPM is insensitive to dopaminergic modulations and (ii) that PD is not related to general deficits in descending pain inhibition beyond the known age-related decline. However, at a trend level, we found differences between PD subtypes (akinetic-rigid, tremor-dominant, mixed) with the strongest impairment of pain inhibition in the akinetic-rigid subtype.

Conclusions

There were no significant differences between CPM responses of patients compared to healthy controls or between patients “on” and “off” medication. Differences between PD subtypes at a trend level point towards different pathophysiological mechanisms underlying the three PD subtypes which warrant further investigation and potentially differential therapeutic strategies in the future.     

Oxidative tailoring of carotenoids: a prospect towards novel functions in plants

Carotenoids not only play a crucial role in their intact form but also are an important reservoir of lipid-derived bioactive mediators. The process is initiated by tailoring enzymes that cleave carotenoids into apocarotenoids. Apocarotenoids act as visual or volatile signals to attract pollinating and seed dispersal agents, and are also key players in allelopathic interactions and plant defense. Recent studies show that the loss of these cleavage enzymes induces the development of axillary branches, indicating that apocarotenoids convey signals that regulate plant architecture. Here, we describe these molecules and the current understanding of their biosynthesis and functions.     

Proteomic analysis of the response of human lung cells to uranium

The industrial use of uranium and particularly of depleted uranium, has pinpointed the need to review its chemical impact on human health. A proteomic approach was used to evaluate the response of a human lung cell line (A549) to uranium. We established the first 2-D reference map of the A549 cell line, identifying 87 spots corresponding to 81 major proteins. Uranium treatment triggered differential expression of 18 spots, of which 14 corresponded to fragments of cytokeratin 8 (CK8) and cytokeratin (CK18) and 1 to peroxiredoxin 1. We probed several hypotheses regarding CK cleavage, and observed that it did not result from caspase or calpain activity. Furthermore, we showed that the fragments are recognised by an anti-ubiquitin antibody (KM691). These results suggest a regulatory pathway involving CK ubiquitinylation or dysfunction in the proteasome-ubiquitin system in response to uranium exposure in human lung cells.     

Nutritional Support from the Intestinal Microbiota Improves Hematopoietic Reconstitution after Bone Marrow Transplantation in Mice

1. Download high-res image (375KB)
2. Download full-size image

     

Micropropagation of Lepidium virginicum (Brassicaceae), a plant with antiprotozoal activity

Summary Micropropagation is a technique to ensure a constant and uniform source of medicinal plants. In this report, we describe the micropropagation of Lepidium virginicum L. (Brassicaceae), a wild plant used as an antiamoebic in traditional Mexican medicine. In vitro-germinated seeds were cultured in Murashige and Skoog (MS) medium to obtain pathogen-free cotyledons, hypocotyls, and apical bud (AB) explants. For induction of morphogenesis, the effect of cytokinins, benzyladenine (BA) and kinetin (KN), combined with auxin, indole-3-acetic acid (IAA) was evaluated. The best rate of shoot proliferation was induced 15 d after culture on MS mineral medium supplemented with IAA∶KN (0.57∶13.94 μM) from AB explants. Maximum shoot elongation was achieved without plant growth regulators. The effect of indole-3-butyric acid (IBA) (14.76 μM) was evaluated for in vitro root induction; 60 d after culture all the shoots had developed roots. All rooted plants were successfully transferred to pots and 100% acclimatized in ex vitro conditions. The methanol extracts from the micropropagated active explants of L. virginicum showed and IC₅₀ antiprotozoal value between 141.90 and 268.53 μg ml⁻¹.     

The ‘Spirit’ of the Thing: The Boundaries of Aboriginal Economic Relations at Australian Common Law

Aboriginal economic relations have been misconstrued as a type of primitive exchange in at least one native title case discussed in this paper. The pursuit by Aboriginal native title claimants of recognition at law of customary economic rights as inherent in, or an adjunct of, native title rights failed in Yarmirr and Others v. Northern Territory of Australia and Others (1998) 156 ALR 370 (the ‘Croker Island case’) for several reasons. The applicant's native title was found to be non‐exclusive of other interests, and a right to trade in resources of the sea was rejected. This case was argued in part by relying on historical material regarding Macassan trading arrangements. The profound alterity of Aboriginal relationships among persons and things, as the Croker Island evidence of property and trade relations demonstrates, have been re‐constituted in legal discourse as an absence of economic relations. In this paper, we argue that there is no sound basis for the distinction made between commercial and non‐commercial native title rights, whether in the Native Title Act 1993 (Commonwealth of Australia), or in recent judicial reasoning. We contend that native title rights and interests constitute a sui generis species of property relations that enable economic rights as conceived in Aboriginal tradition and custom to circulate in the modern market. Aboriginal customary economic relations of and between Aboriginal groupings are markedly distinct from, yet not incommensurable with, the normative conception of economic relations in the Australian market. We argue that a reformulation of the current Australian legal ideas about economic life is necessary for the recognition of Aboriginal economic institutions in native title claims and other economic arenas.