FlyGEM, a full transcriptome array platform for the Drosophila community

We have constructed a DNA microarray to monitor expression of predicted genes in Drosophila. By using homotypic hybridizations, we show that the array performs reproducibly, that dye effects are minimal, and that array results agree with systematic northern blotting. The array gene list has been extensively annotated and linked-out to other databases. Incyte and the NIH have made the platform available to the community via academic microarray facilities selected by an NIH committee.     

The immunoregulatory and allergy-associated cytokines in the aetiology of the otitis media with effusion

Inflammation in the middle ear mucosa, which can be provoked by different primary factors such as bacterial and viral infection, local allergic reactions and reflux, is the crucial event in the pathogenesis of otitis media with effusion (OME). Unresolved acute inflammatory responses or defective immunoregulation of middle inflammation can promote chronic inflammatory processes and stimulate the chronic condition of OME. Cytokines are the central molecular regulators of middle ear inflammation and can switch the acute phase of inflammation in the chronic stage and induce molecular-pathological processes leading to the histopathological changes accompanying OME. In this review we present cytokines identified in otitis media, immunoregulatory [interleukin (IL)-2, IL-10, transforming growth factor-beta]) and allergy associated (IL-4, IL-5, granulocyte-macrophage colony-stimulating factor), as crucial molecular regulators, responsible for chronic inflammation in the middle ear and the chronic condition of OME.     

Molecular phylogeny of two lineages of Leuciscinae cyprinids (Telestes and Scardinius) from the peri-Mediterranean area based on cytochrome b data

We examined phylogenetic relationships in two lineages of Leuciscinae cyprinid fishes based on the sequence data of the complete mitochondrial DNA region coding for the cytochrome b gene (1140 bp). Telestes includes obligate riverine, moderately cold water-adapted species whereas Scardinius comprises warm-adapted species living in lowland lakes and still waters of rivers and streams. We also analysed selected representatives of Leuciscus and Phoxinellus because the taxonomic status of some species belonging to these genera is dubious and they could be placed in the genus Telestes. The study includes 18 species, 43 populations, and 111 individuals from 9 of the 14 peri-Mediterranean ichthyogeographic districts. Clades recovered from the phylogenetic analyses do not support previous taxonomic assumptions based on morphology. Telestes, Leuciscus, and Phoxinellus do not form monophyletic assemblages; phylogenetic analyses suggest that L. polylepis, L. turskyi, P. croaticus, and P. metohiensis should be included in Telestes. Similarly, populations of Scardinius erythrophthalmus do not cluster together and the endangered S. scardafa, endemic to central Italy and surviving in a single locality, is nested within them. The radiations of Telestes and Scardinius occurred in different time periods. A major diversification of Telestes is consistent with a sea dispersal during the freshwater Messinian "Lago Mare" phase of the Mediterranean Sea. Cladogenetic events within Scardinius are likely related to the extension and confluence of river drainages in lowlands following multiple lowering of the sea level during the Quaternary glaciations.     

Strategies for structural proteomics of prokaryotes: Quantifying the advantages of studying orthologous proteins and of using both NMR and X-ray crystallography approaches

Only about half of non-membrane-bound proteins encoded by either bacterial or archaeal genomes are soluble when expressed in Escherichia coli (Yee et al., Proc Natl Acad Sci USA 2002;99:1825-1830; Christendat et al., Prog Biophys Mol Biol 200;73:339-345). This property limits genome-scale functional and structural proteomics studies, which depend on having a recombinant, soluble version of each protein. An emerging strategy to increase the probability of deriving a soluble derivative of a protein is to study different sequence homologues of the same protein, including representatives from thermophilic organisms, based on the assumption that the stability of these proteins will facilitate structural analysis. To estimate the relative merits of this strategy, we compared the recombinant expression, solubility, and suitability for structural analysis by NMR and/or X-ray crystallography for 68 pairs of homologous proteins from E. coli and Thermotoga maritima. A sample suitable for structural studies was obtained for 62 of the 68 pairs of homologs under standardized growth and purification procedures. Fourteen (eight E. coli and six T. maritima proteins) samples generated NMR spectra of a quality suitable for structure determination and 30 (14 E. coli and 16 T. maritima proteins) samples formed crystals. Only three (one E. coli and two T. maritima proteins) samples both crystallized and had excellent NMR properties. The conclusions from this work are: (1) The inclusion of even a single ortholog of a target protein increases the number of samples for structural studies almost twofold; (2) there was no clear advantage to the use of thermophilic proteins to generate samples for structural studies; and (3) for the small proteins analyzed here, the use of both NMR and crystallography approaches almost doubled the number of samples for structural studies.     

Role of apoptosis and Fas/FasL system in the oogenesis of the spotted ray Torpedo marmorata

In the present paper we investigated the role played by apoptosis during oogenesis in the cartilaginous fish Torpedo marmorata. TEM, TUNEL and immunohistochemical techniques were employed to specifically reveal morphological and biochemical hallmarks of apoptosis in specimens from birth to sexual maturity. Data obtained demonstrate that apoptosis occurs in prefollicular oocyte selection, in maintaining the homeostasis of granulosa in healthy growing oocyte and in resorbing atretic follicles. In this respect, the involvement of apoptosis in Torpedo marmorata oogenesis closely parallels that found in mammals, thus confirming that strategies of germ cell selection among vertebrates have been evolutionarily preserved.     

Decorin reverses the repressive effect of autocrine-produced TGF-beta on mouse macrophage activation

Several cytokines or growth factors induce macrophages to proliferate, become activated, differentiate, or die through apoptosis. Like the major macrophage activator IFN-gamma, the extracellular matrix protein decorin inhibits proliferation and protects macrophages from the induction of apoptosis. Decorin enhances the IFN-gamma-induced expression of the IAalpha and IAbeta MHC class II genes. Moreover, it increases the IFN-gamma- or LPS-induced expression of inducible NO synthase, TNF-alpha, IL-1beta, and IL-6 genes and the secretion of these cytokines. Using a number of extracellular matrix proteins, we found a negative correlation between adhesion and proliferation. However, the effects of decorin on macrophage activation do not seem to be mediated through its effect on adhesion or proliferation. Instead, this proteoglycan abolishes the binding of TGF-beta to macrophages, as shown by Scatchard analysis of (125)I-labeled TGF-beta, which, in the absence of decorin, showed a K(d) of 0.11 +/- 0.03 nM and approximately 5000 receptors/cell. This was confirmed when we treated macrophages with Abs to block the endogenously produced TGF-beta, which enhanced macrophage activation in a way similar to decorin. The increase in activation mediated by decorin demonstrates that macrophages are under negative regulation that can be reversed by proteins of the extracellular matrix.     

Plasma distribution of apoA-IV in patients with coronary artery disease and healthy controls

Recent studies showed lower apolipoprotein A-IV (apoA-IV) plasma concentrations in patients with coronary artery disease (CAD). The actual distribution of the antiatherogenic apoA-IV in human plasma, however, is discussed controversially and it was never investigated in CAD patients. We therefore developed a gentle technique to separate the various apoA-IV-containing plasma fractions. Using a combination of precipitation of all lipoproteins with 40% phosphotungstic acid and 4 M MgCl2, as well as immunoprecipitation of all apoA-I-containing particles with an anti-apoA-I antibody, we obtained three fractions of apoA-IV: lipid-free apoA-IV (about 4% of total apoA-IV), apoA-IV associated with apoA-I (LpA-I:A-IV, 12%), and apoA-I-unbound but lipoprotein-containing apoA-IV (LpA-IV, 84%). We compared these three apoA-IV fractions between 52 patients with a history of CAD and 52 age- and sex-matched healthy controls. Patients had significantly lower apoA-IV levels when compared to controls (10.28 +/- 3.67 mg/dl vs. 11.85 +/- 2.82 mg/dl, P = 0.029), but no major differences for the three plasma apoA-IV fractions. We conclude that our gentle separation method reveals a different distribution of apoA-IV than in many earlier studies. No major differences exist in the apoA-IV plasma distribution pattern between CAD patients and controls. Therefore, the antiatherogenic effect of apoA-IV has to be explained by other functional properties of apoA-IV (e.g., the antioxidative characteristics).     

Expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins

Much effort has been dedicated to the design of significantly red shifted variants of the green fluorescent protein (GFP) from Aequoria victora (av). These approaches have been based on classical engineering with the 20 canonical amino acids. We report here an expansion of these efforts by incorporation of an amino substituted variant of tryptophan into the "cyan" GFP mutant, which turned it into a "gold" variant. This variant possesses a red shift in emission unprecedented for any avFP, similar to "red" FPs, but with enhanced stability and a very low aggregation tendency. An increasing number of non-natural amino acids are available for chromophore redesign (by engineering of the genetic code) and enable new general strategies to generate novel classes of tailor-made GFP proteins.     

TCR comodulation of nonengaged TCR takes place by a protein kinase C and CD3 gamma di-leucine-based motif-dependent mechanism

One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Ti beta chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (K(D) = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (K(D) = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.