DNA-based methods for the detection and the identification of phytoplasmas in insect vector extracts

DNA extraction and storage methods have been evaluated with laboratory-reared leafhoppers and/or field-collected leafhoppers and psyllids. Detection of four different phytopathogenic phytoplasmas, belonging to three taxonomic groups, has been achieved by several direct or nested polymerase chain reaction (PCR) methods with such DNA extracts. Reactions differed in both the 16/23S ribosomal primer pairs used and the specific assay and cycling conditions. Merits and possible hindrances of the various primer pairs, in relation to insect DNA extracts, are discussed. However, identification of the phytoplasma(s) necessarily relied on comparison of the polymorphism in length of the amplified DNA fragments obtained by restriction with appropriate endonucleases. Endonuclease digestion is crucial for determining the identity (subgroup affiliation) of phytoplasmas of the same groups that can be carried by an individual vector.     

Splicing signals and factors in plant intron removal

Constitutive splicing of the potato invertase mini-exon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5' splice site of the adjacent intron and a U(11) element found just downstream of the branchpoint in the upstream intron [Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422-433]. The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences. Plant introns differ from their vertebrate and yeast counterparts in being UA- or U-rich (up to 85% UA). One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins. We are adopting three approaches to studying the RNA-protein interactions in plant splicing. First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants. Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts. Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened.     

Inhibition of somatostatin receptor 5-signaling by mammalian regulators of G-protein signaling (RGS) in yeast

Regulators of G-protein signaling (RGSs) are negative regulators of G-protein coupled receptor (GPCR)-mediated signaling that function to limit the lifetime of receptor-activated G(alpha)-proteins. Here we show that four mammalian RGSs differentially inhibit the activation of a FUS1--LacZ reporter gene by the STE2 encoded GPCR in yeast. In order to examine the role of the GPCR in modulating RGS function, we functionally expressed the human somatostatin receptor 5 (SST(5)) in yeast. In the absence of RGSs, FUS1--LacZ activation in response to somatostatin increased in a dose-dependent manner in cells expressing SST(5). In contrast to the results obtained with Ste2p, all RGSs completely inhibited SST(5)-mediated signaling even at concentrations of agonist as high as 10(minus sign5) M. The ability of RGSs to inhibit SST(5) signaling was further assessed in cells expressing modified Gpa1 proteins. Even though SST(5)-mediated FUS1--LacZ activation was 5-fold more efficient with a Gpa1p/G(i3alpha) chimera, response to somatostatin was completely abolished by all four RGSs. Furthermore, we demonstrate that RGS1, RGS2 and RGS5 have reduced ability to inhibit SST(5)-mediated activation of the RGS-resistant Gpa1p(Gly302Ser) mutant suggesting that the ability to interact with the G(alpha)-protein is required for the inhibition of signaling. Taken together, our results indicate that RGSs serve as better GAPs for Gpa1p when activated by SST(5) than when this G-protein is activated by Ste2p.     

Population dynamics of Cacopsylla melanoneura (Homoptera: Psyllidae), a vector of apple proliferation phytoplasma in northwestern Italy

Apple proliferation is a phytoplasma-associated disease transmitted by insects causing serious damage and economic losses to apple orchards. Investigations were carried out in 1999 and 2000 in northwestern Italy to identify the vector of apple proliferation and to study its population dynamics. Yellow sticky traps and beat tray samples revealed the presence of the psyllid Cacopsylla melanoneura (Forster) in eight apple orchards in the Aosta Valley. The species completes one generation per year; the overwintered psyllids colonized apple trees beginning in late January, whereas the springtime generation was observed beginning in early May. The offspring adults remained in apple orchards until the end of June, when they began to move onto other hosts. During 1999 and 2000, all apple trees present in the investigated orchards were visually checked to assess the fluctuation of disease symptoms. Polymerase chain reaction and restriction fragment-length polymorphism confirmed the presence of the apple proliferation phytoplasmas in both overwintering and offspring insects as well as in symptomatic apple plants. The ability of C. melanoneura to vector the disease was assessed by preliminary transmission trials. Overwintered psyllids, collected in the most affected orchards, caged on healthy apple test plants transmitted apple proliferation phytoplasmas.     

The telomeric region of BTA18 containing a potential QTL region for health in cattle exhibits high similarity to the HSA19q region in humans

We have applied a targeted physical mapping approach, based on the isolation of bovine region-specific large-insert clones using homologous human sequences and chromosome microdissection, to enhance the physical gene map of the telomeric region of BTA18 and to prove its evolutionary conservation. The latter is a prerequisite to exploit the dense human gene map for future positional cloning approaches. Partial sequencing and homology search were used to characterize 20 BACs targeted to the BTA18q2.4-q2.6 region. We used fluorescence in situ hybridization (FISH) to create physical maps of 11 BACs containing 15 gene loci; these BACs served as anchor loci. Using these approaches, 12 new gene loci (CKM, STK13, PSCD2, IRF3, VASP, ACTN4, ITPKC, CYP2B6, FOSB, DMPK, MIA, SIX5) were assigned on BTA18 in the bovine cytogenetic map. A resolved physical map of BTA18q2.4-q2.6 was developed, which encompasses 28 marker loci and a comparative cytogenetic map that contains 15 genes. The mapping results demonstrate the high evolutionary conservation between the telomeric region of BTA18q and HSA19q.     

Functional and structural characterization of anti-β1-adrenoceptor autoantibodies of spontaneously hypertensive rats

Eighteen month old spontaneously hypertensive rats (SHR-rats) showed myocardial dysfunction and autoantibodies directed against the ₁-adrenoceptor similarly as known in human dilated cardiomyopathy or Chagas' disease. The agonist-like antibodies were able to activate the ₁-adrenoceptor mediated signal transduction cascade in cultured rat cardiomyocytes and induced a long-lasting stimulatory effect resulting in a harmful adrenergic overdrive. The antibodies recognized an epitope of the second extracellular loop of the ₁-adrenoceptor identical to that epitope identified in Chagas' disease. In conclusion, our assumption is supported that old SHR-rat are an useful animal model for investigating the role of anti-₁-adrenoceptor antibodies in the induction of human cardiomyopathy.     

Expression of α1-adrenergic receptor subtypes in heart cell culture

Three ₁AR subtypes have been cloned so far and are designated as _1a, _1b, and _1d. Organspecific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of ₁AR subtypes on RNA and proteinlevel in heart tissue, cultured cardiomyocytes and nonmyocytes of the rat. Each ₁ARsubtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the _1aAR subtype is preferentially expressed in adult cardiomyocytes, the _1bAR subtype was preferentially expressed in the nonmyocyte cell fraction. The RTPCR results were confirmed by Westernblotting (_1b) and immunocytochemical studies. Incubation with an ₁agonist (phenylephrine) for 72 h led to a significant reduction of the _1bAR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an ₁antagonist (prazosin) induced a 1.6 fold upregulation of the _1aAR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the ₁AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents.     

iNKT cells require CCR4 to localize to the airways and to induce airway hyperreactivity

iNKT cells are required for the induction of airway hyperreactivity (AHR), a cardinal feature of asthma, but how iNKT cells traffic to the lungs to induce AHR has not been previously studied. Using several models of asthma, we demonstrated that iNKT cells required the chemokine receptor CCR4 for pulmonary localization and for the induction of AHR. In both allergen-induced and glycolipid-induced models of AHR, wild-type but not CCR4-/- mice developed AHR. Furthermore, adoptive transfer of wild-type but not CCR4-/- iNKT cells reconstituted AHR in iNKT cell-deficient mice. Moreover, we specifically tracked CCR4-/- vs wild-type iNKT cells in CCR4-/-:wild-type mixed BM chimeric mice in the resting state, and when AHR was induced by protein allergen or glycolipid. Using this unique model, we showed that both iNKT cells and conventional T cells required CCR4 for competitive localization into the bronchoalveolar lavage/airways compartment. These results establish for the first time that the pulmonary localization of iNKT cells critical for the induction of AHR requires CCR4 expression by iNKT cells.