Expression of Green Fluorescent Protein in <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> Is Affected by Passage Through Host Plants

Xylella fastidiosa, a Gram-negative bacterial plant pathogen, causes Pierces disease of grapevine in North America. In South America the pathogen causes citrus variegated chlorosis, which is widespread in Brazil. We have introduced into Xylella fastidiosa a mini-Tn5 transposon that encodes a green fluorescent protein (GFP) gene optimized for expression in bacteria. The mini-Tn5 derivative was inserted into different sites of the genome in independent transconjugants as determined by Southern blotting. The GFP gene was expressed well and to different levels in different transconjugants. Four independent transconjugants were separately used to inoculate sweet orange and tobacco seedlings. The transconjugants were able to colonize the plants and were subsequently isolated from points distal to the inoculation sites. When the relative fluorescence of the transconjugants that had been passed through either tobacco or sweet orange was compared with that of the same transconjugant maintained continuously in vitro, we observed that passage through either plant host significantly increased the level of expression of the GFP. The increased level of expression of GFP was transient, and was lost upon further culture in vitro. Xylella fastidiosa forms biofilms in planta which are believed to represent a metabolically differentiated state. The increased expression of GFP observed after passage through plants may be accounted for by this phenomenon.     

Proteomic approach: Identification of <Emphasis Type="Italic">Medicago truncatula</Emphasis> proteins induced in roots after infection with the pathogenic oomycete <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>

The legume root rot disease caused by the oomycete pathogen Aphanomyces euteiches is one major yield reducing factor in legume crop production. A comparative proteomic approach was carried out in order to identify proteins of the model legume Medicago truncatula which are regulated after an infection with A. euteiches. Several proteins were identified by two dimensional gel electrophoresis to be differentially expressed after pathogen challenge. Densitometric evaluation of expression values showed different regulation during the time-course analysed. Proteins regulated during the infection were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Among the differentially expressed proteins, two encoded putative cell wall proteins and two were designated as small heat shock proteins. Furthermore, an isoform of the chalcone-O-methyltransferase was found to be increased in infected roots. The majority of induced proteins belonged to the family of class 10 of pathogenesis related proteins (PR10). Previously, various PR10-like proteins have been shown to be regulated by general stress or abscisic acid (ABA). Therefore, these proteins were further investigated concerning their regulation in response to drought stress and exogenous ABA-application. Complex regulation patterns were identified: three of the A. euteiches-induced PR10-like proteins were also induced by exogenous ABA- but none of them is induced after drought stress. In contrast, three of these proteins are down-regulated by drought stress. Hence, the strong expression of different PR10-family members and their regulation profiles indicates that this set of proteins plays a major role during root adaptations to various stress conditions.     

Anti-Ri antibodies associated with short-term memory deficits and a mature cystic teratoma of the ovary

BACKGROUND: The IgG autoantibody ANNA-2 (anti-Ri) is a type 2 antineuronal antibody that has been found to bind to highly conserved and widely distributed adult brain proteins encoded by the Nova-1 and Nova-2 genes. Anti-Ri antibodies are typically detected in the serum and cerebrospinal fluids of patients with neurological disorders such as opsoclonus/myoclonus and cerebellar ataxia and in association with gynecologic and breast malignancies. CASE PRESENTATION: This report describes an unusual example of a 33-year-old female patient who developed short-term memory deficits over a 3-month period. An extensive neurological work-up, including a panel of paraneoplastic markers was negative with the exception of a high titer serum Anti-Ri (1:15,3600). A large left ovarian mass was palpated, surgically resected and eventually diagnosed as a mature cystic teratoma. Post-operatively, memory deficits had disappeared within 1 month and serum Anti-Ri titers had decreased significantly to 1:256. An extensive diagnostic work-up for other malignancies was negative. CONCLUSION: Although, Anti-Ri antibodies are typically associated with malignancies, this case illustrates the potential association between benign tumors and this autoantibody.     

Characterization of bradykinin receptors in canine cultured corneal epithelial cells: Pharmacological and functional studies

The pharmacological properties of bradykinin (BK) receptors were characterized in canine cultured corneal epithelial cells (CECs) using [(3)H]-BK as a radioligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant of 0.34 +/- 0.07 nM and a maximum receptor density of 179 +/- 23 fmol/mg protein. Neither a B(1) receptor-selective agonist (des-Arg(9)-BK) nor antagonist ([Leu(8), des-Arg(9)]-BK) significantly inhibited [(3)H]-BK binding to CECs, thus excluding the presence of B(1) receptors in canine CECs. The specific binding of [(3)H]-BK to CECs was inhibited by B(2) receptor-selective agonists (BK and kallidin) and antagonists (Hoe 140 and [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK), with a best fit using a one-binding-site model. The order of potency for the inhibition of [(3)H]-BK binding was BK = Hoe 140 > kallidin > [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK. Stimulation of CECs by BK produced a concentration-dependent accumulation of inositol phosphates (IP) and an initial transient peak of intracellular Ca(2+). B(2) receptor-selective antagonist ([D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK) significantly antagonized the BK-induced responses with dissociation constants of 6.0-6.1. Pretreatment of CECs with pertussis toxin (PTX) or cholera toxin did not alter the BK-induced IP accumulation. Incubation of CECs in the absence of external Ca(2+) led to a significant attenuation of the IP accumulation induced by BK. These results demonstrate that BK directly stimulates phospholipase C-mediated signal transduction through BK B(2) receptors via a PTX-insensitive G protein in canine CECs. This effect may function as the transducing mechanism for BK-mediated cellular responses.     

When and where was the most recent common ancestor?

 The structured coalescent is investigated for single-locus, digenic samples in the diffusion limit of the unidimensional stepping-stone model for homogeneous, isotropic migration and random genetic drift. Let T denote the scaled time to the most recent common ancestor (MRCA) of the two genes, and let Z designate the scaled deviation of the position of the MRCA from the average position of the two genes. The joint probability density of T and Z is evaluated explicitly. Both the marginal and conditional distributions of T have infinite expectation, as does the marginal distribution of Z. Conditioned on T = τ, the distribution of Z is Gaussian with mean zero and variance 2τ. The main results are extended to anisotropic migration. The results establish the existence of and define in the diffusion limit a retrospective stochastic process for digenic samples in one spatial dimension. Received: 1 May 2001 / Revised version: 2 September 2001 / Published online: 8 February 2002     

Transformation in Heterokaryons of <Emphasis Type="Italic">Neurospora crassa</Emphasis> Is Nuclear Rather Than Cellular Phenomenon

In Neurospora crassa, multinucleate macroconidia are used for genetic transformation. The barrier for such a transformation can be either at the cell membrane level or at the nuclear membrane level. For assessment of these possibilities, a forced heterokaryon (containing two genetically marked nuclei and auxotrophic for histidine) of Neurospora crassa was transformed with a plasmid containing his-3 ⁺ gene. The transformants, which could grow without histidine supplementation, were then resolved into component homokaryons to determine into which nucleus or nuclei the plasmid had entered. Our results suggest that the barrier for transformation in Neurospora crassa is at the nuclear level, not at the cell membrane level. In a heterokaryon containing two genetically distinct nuclei, plasmid DNA integrated into only one of the nuclear types at any instance, but never into both nuclear types. Thus, in Neurospora crassa, the competent nucleus is essential for the transformation event to take place, and at a given time only one type of nucleus is competent to take up the exogenous DNA. Genomic Southern analysis showed that the transformants harbor both ectopic and homologous integrations of the plasmid DNA. The type and number of integrations were reflected at the post-translational level, since the specific activity of histidinol dehydrogenase (the translation product of his-3 ⁺ gene) was variable among several transformants and always less than the level of the wild type. Received: 24 July 2001 / Accepted: 15 August 2001     

<Emphasis Type="Italic">Mariner</Emphasis>-like elements in <Emphasis Type="Italic">Rhynchosciara americana</Emphasis> (Sciaridae) genome: molecular and cytological aspects

Two mariner-like elements, Ramar1 and Ramar2, are described in the genome of Rhynchosciara americana, whose nucleotide consensus sequences were derived from multiple defective copies containing deletions, frame shifts and stop codons. Ramar1 contains several conserved amino acid blocks which were identified, including a specific D,D(34)D signature motif. Ramar2 is a defective mariner-like element, which contains a deletion overlapping in most of the internal region of the transposase ORF while its extremities remain intact. Predicted transposase sequences demonstrated that Ramar1 and Ramar2 phylogenetically present high identity to mariner-like elements of mauritiana subfamily. Southern blot analysis indicated that Ramar1 is widely represented in the genome of Rhynchosciara americana. In situ hybridizations showed Ramar1 localized in several chromosome regions, mainly in pericentromeric heterochromatin and their boundaries, while Ramar2 appeared as a single band in chromosome A.     

Chemical communication in mating behaviour of the slave-making ant <Emphasis Type="Italic">Polyergus rufescens</Emphasis> (Hymenoptera,Formicidae): 3-ethyl-4-methylpentanol as a critical component of the queen sex pheromone

The aim of the research reported here was to determine whether 3-ethyl-4-methylpentanol, a minor but crucial component of the sex pheromone of the North American slave-making ant species Polyergus breviceps, was also a component of the sex pheromone of the European congener Polyergus rufescens. Thus, the contents of mandibular glands of P. rufescens virgin queen were extracted and analysed. The main component of the extracts was methyl 6-methylsalicylate and 3-ethyl-4-methylpentanol was identified as one of several minor components. Further analyses showed that the insects produce mainly the (R)-enantiomer of the alcohol. Males’ responses to various blends of methyl 6-methylsalicylate with the racemate or the pure enantiomers of 3-ethyl-4- methylpentanol were tested in field behavioural bioassays. The data showed that blends of methyl 6- methylsalicylate and 3-ethyl-4-methylpentanol were strongly synergistic, with the most active ratios being biased toward the first component. The addition of other minor components to the binary blend neither increased nor decreased responses by males. Only the (R)-enantiomer of the alcohol was biologically active; its antipode did not inhibit attraction. The results are discussed in terms of the evolution of signals, and are compared with the results previously obtained for the allopatric species Polyergus breviceps. Received 12 November 2007; revised 10 January 2008; accepted 11 January 2008.     

<Emphasis Type="Italic">Podospora anserina</Emphasis>: a model organism to study mechanisms of healthy ageing

The filamentous ascomycete Podospora anserina has been extensively studied as an experimental ageing model for more than 50 years. As a result, a huge body of data has been accumulated and various molecular pathways have been identified as part of a molecular network involved in the control of ageing and life span. The aim of this review is to summarize data on P. anserina ageing, including aspects like respiration, cellular copper homeostasis, mitochondrial DNA (mtDNA) stability/instability, mitochondrial dynamics, apoptosis, translation efficiency and pathways directed against oxidative stress. It becomes clear that manipulation of several of these pathways bears the potential to extend the healthy period of time, the health span, within the life time of the fungus. Here we put special attention on recent work aimed to identify and characterize this type of long-lived P. anserina mutants. The study of the molecular pathways which are modified in these mutants can be expected to provide important clues for the elucidation of the mechanistic basis of this type of 'healthy ageing' at the organism level.