Differential requirement for STAT by gain-of-function and wild-type receptor tyrosine kinase Torso in Drosophila

Malignant transformation frequently involves aberrant signaling from receptor tyrosine kinases (RTKs). These receptors commonly activate Ras/Raf/MEK/MAPK signaling but when overactivated can also induce the JAK/STAT pathway, originally identified as the signaling cascade downstream of cytokine receptors. Inappropriate activation of STAT has been found in many human cancers. However, the contribution of the JAK/STAT pathway in RTK signaling remains unclear. We have investigated the requirement of the JAK/STAT pathway for signaling by wild-type and mutant forms of the RTK Torso (Tor) using a genetic approach in DROSOPHILA: Our results indicate that the JAK/STAT pathway plays little or no role in signaling by wild-type Tor. In contrast, we find that STAT, encoded by marelle (mrl; DStat92E), is essential for the gain-of-function mutant Tor (Tor(GOF)) to activate ectopic gene expression. Our findings indicate that the Ras/Raf/MEK/MAPK signaling pathway is sufficient to mediate the normal functions of wild-type RTK, whereas the effects of gain-of-function mutant RTK additionally require STAT activation.     

Synthesis,characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels

Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In this report, we determine the three-dimensional structure of HgTX(1) using NMR spectroscopy (PDB-code: 1HLY). HgTX(1) was found to possess a structure similar to previously characterized K+ channel toxins (e.g. margatoxin) consisting of a three-stranded antiparallel beta-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 3(10) helix and part alpha helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interaction with the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX(1). On the basis of previous studies (see above) and our NMR data, a HgTX(1) mutant (HgTX(1)-A19C) was engineered, expressed, and purified. HgTX(1)-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-, and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX(1)-A19C in radioligand binding studies indicated that these hongotoxin(1) analogues retain high-affinity for voltage-gated K+ channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin(1) analogues were used to investigate the localization of K+ channels in brain sections. The distribution of toxin binding closely follows the distribution of K(v)1.2 immunoreactivity with the highest expression levels in the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescently labeled HgTX(1) analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.     

An european pupil project linked to the scientific aims of the experiment AQUARIUS-XENOPUS on the taxi Soyuz flight Andromede to ISS

The German-French biological experiment AQUARIUS-XENOPUS which flew on the Soyuz flight Andromede to the International Space Station ISS (launched October 21, 2001 in Baikonour/Kazakhstan) was extended by an outreach project. Pupils of class 10 to 12 from Ulm/D and Nancy-Tomblaine/F studied swimming behavior of Xenopus tadpoles on ground. They were instructed to perform all experimental steps following the protocol of similar video recordings on ISS. After the flight, they evaluated the kinetics of swimming of both ground controls and space animals. The pupil project included theoretical components to introduce them to the field of gravitational biology. One feature of the project was the exchange of ideas between pupils by meetings which took place in Ulm (June 2001), Nancy (February 2002) and Paris (May 2002). We consider our approach as a successful way to include young people in space experiments on a cheap cost level and to bring ideas of gravitational biology into the curricula of European schools.     

Oxidized low-density lipoprotein-induced apoptosis

Cultured cells are able to oxidize low-density lipoproteins (LDL) and oxidized LDL (oxLDL), which are present in atherosclerosis areas, exhibit a variety of biological properties potentially involved in atherogenesis. This review is focused on the toxicity of oxLDL, more precisely on the toxic compounds generated during LDL oxidation, the features and the mechanisms of cell death (apoptosis or necrosis) induced by oxLDL. After internalization, toxic oxidized lipids, namely lipid peroxides, oxysterols and aldehydes, induce modifications of cell proteins, elicit oxidative stress, lipid peroxidation and alter various signaling pathways and gene expression. These events may participate in the toxic effect, and converge to trigger an intense, delayed and sustained calcium peak which elicits either apoptosis or necrosis processes. OxLDL-induced apoptosis involves both mitochondrial and death-receptor (Fas/FasL) apoptotic pathways, thereby activating the classical caspase cascade and subsequent biochemical and morphological apoptotic features. When apoptosis is blocked by overexpression of Bcl-2, oxLDL trigger necrosis through a calcium-dependent pathway. Apoptosis occurring in atherosclerotic areas is potentially involved in endothelial cell lining defects, necrotic core formation and plaque rupture or erosion which may trigger atherothrombotic events. However, the precise role of oxLDL in apoptosis/necrosis occurring in vivo in atherosclerotic plaques remains to be clarified.     

Differential carbohydrate metabolism conducts morphogenesis in embryogenic callus of Hevea brasiliensis (Müll. Arg.)

Somatic embryogenesis in Hevea is stimulated when the embryogenesis induction medium contains maltose, rather than glucose, fructose, or sucrose, in equimolarity (Blanc et al., 1999). Kinetic analyses were carried out on various physiological and biochemical indicators over the 8 weeks that the induction phase then expression of somatic embryogenesis can take. Embryogenesis induction in the presence of glucose, fructose or sucrose revealed strong callus growth in the first 3-4 weeks, associated with a high intra- and extracellular hexose content, a high starch content and a substantial decline in protein synthesis. In the presence of maltose, callus growth was slow and only half that seen with sucrose. This morphogenetic behaviour is associated with a drop in endogenous hexose and starch contents, and an increase in protein synthesis in the first three weeks of culture. The induction of embryogenesis in the presence of maltose was uniform and twice as fast as with sucrose supply. At the end of culture, peroxidase activity, antioxidant and membrane protein contents increased in these calluses; these characteristics may be associated with somatic embryo organization and with the maintenance of effective membrane integrity within a nutrient environment that has become limiting. These new results tally with data in the literature on the roles of sugars, and provide some precise information with regard to the 'carbohydrate deficit' hypothesis usually put forward to explain maltose action. An analysis of these results led to the hypothesis that regulation of endogenous hexose contents at a low level, through slow maltose hydrolysis, was a key element of the biochemical signal leading this callus towards somatic embryogenesis.     

Characterization of four lectin-like receptor kinases expressed in roots of Medicago truncatula. Structure, location, regulation of expression, and potential role in the symbiosis with Sinorhizobium meliloti

To study the role of LecRK (lectin-like receptor kinase) genes in the legumerhizobia symbiosis, we have characterized the four Medicago truncatula Gaernt. LecRK genes that are most highly expressed in roots. Three of these genes, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, encode proteins most closely related to the Class A LecRKs of Arabidopsis, whereas the protein encoded by the fourth gene, MtLecRK1;1, is most similar to a Class B Arabidopsis LecRK. All four genes show a strongly enhanced root expression, and detailed studies on MtLecRK1;1 and MtLecRK7;2 revealed that the levels of their mRNAs are increased by nitrogen starvation and transiently repressed after either rhizobial inoculation or addition of lipochitooligosaccharidic Nod factors. Studies of the MtLecRK1;1 and MtLecRK7;2 proteins, using green fluorescent protein fusions in transgenic M. truncatula roots, revealed that they are located in the plasma membrane and that their central transmembrane-spanning helix is required for correct sorting. Moreover, their lectin-like domains appear to be highly glycosylated. Of the four proteins, only MtLecRK1;1 shows a high conservation of key residues implicated in monosaccharide binding, and molecular modeling revealed that this protein may be capable of interacting with Nod factors. However, no increase in Nod factor binding was found in roots overexpressing a fusion in which the kinase domain of this protein had been replaced with green fluorescent protein. Roots expressing this fusion protein however showed an increase in nodule number, suggesting that expression of MtLecRK1;1 influences nodulation. The potential role of LecRKs in the legume-rhizobia symbiosis is discussed.     

The expanding interleukin-1 family and its receptors: do alternative IL-1 receptor/signaling pathways exist in the brain?

Interleukin-1 (IL-1) has been implicated in neuroimmune responses and has pleiotropic actions in the brain. Compelling evidence has shown that IL-1 is a major mediator of inflammation and the progression of cell death in response to brain injury and cerebral ischemia. Its expression is strongly increased in these pathological conditions, and central administration of exogenous IL-1 significantly exacerbates ischemic brain damage. In contrast, inhibiting IL-1 actions (by intracerebroventricular [icv] injection of IL-1ra, neutralizing antibody to IL-1 or caspase-1 inhibitor) significantly reduces ischemic brain damage. IL-1 acts by binding to the IL-1 type-1 receptor (IL-1RI), which is to date, the only known functional receptor for IL-1. However, our recent investigations suggest that IL-1 can act independently of IL-1RI, raising the possibility that additional, as yet undiscovered, receptor(s) for IL-1 exist in the brain. The recent characterization of putative, new IL-1 ligands and new IL-1 receptor-related molecules leads to the hypothesis that there might be alternative IL-1 signaling pathway(s) in the central nervous system (CNS).     

A new mouse limb mutation identifies a <Emphasis Type="Italic">Twist</Emphasis> allele that requires interacting loci on Chromosome 4 for its phenotypic expression

Pluridigite (Pdt) is a semi-dominant mutation obtained after a mutagenesis experiment with ethyl-nitroso-urea (ENU). The mutant exhibits abnormal skeletal pattern formation characterized by the formation of extra digits (polydactyly) in the preaxial (anterior) part of the hindlimbs. The phenotype shows incomplete penetrance, depending on the genetic background. In an F₂ cross with C57BL/6, the phenotype could not be associated with a single locus. Strong linkage was observed with markers located on Chromosome (Chr) 12, in a 2-cM interval between D12Mit136 and D12Mit153. This region contains the Twist gene, and we show that the [Pdt] phenotype is dependent upon a new allele of Twist. We further identified that the whole Chr 4 is associated with the [Pdt] phenotype. The Pluridigite phenotype thus results from the combination of a Twist mutant allele and at least two additional loci.     

HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line

The regulation of intercellular adhesion by hepatocyte growth factor (HGF) was examined on a novel nontumorigenic gastric epithelial cell line (IMGE-5) derived from H-2Kb-tsA58 transgenic mice. IMGE-5 cells constitutively expressed cytokeratin 18 and HGF receptors. Under permissive conditions (33 degrees C + interferon-gamma), IMGE-5 cells proliferated rapidly but did not display membrane expression of adherens and tight junction proteins. Under nonpermissive conditions, their proliferation was decreased and they displayed a strong, localized membrane expression of E-cadherin/beta-catenin and occludin/ZO-1. HGF treatment largely prevented the targeting of ZO-1 to the tight junction and induced a significant decrease of the transepithelial resistance measured across a confluent IMGE-5 cell monolayer. HGF rapidly increased the tyrosine phosphorylation of ZO-1 and decreased its association with occludin in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. PI 3-kinase was also involved in HGF-induced migration of IMGE-5 cells. Our results demonstrate that 1) HGF prevents the appearance of ZO-1 in the membrane during epithelial cell differentiation; 2) HGF causes partial relocalization of ZO-1 to the cytoplasm and nucleus and concomitantly stimulates cell dissociation and migration; and 3) IMGE-5 cells offer a useful model for the study of gastric epithelial cell differentiation.     

Determination of the copy number of the nuclear rDNA and beta-tubulin genes of Pneumocystis carinii f. sp. hominis using PCR multicompetitors

Multiple copies of a gene may lead to difficulty in the interpretation of typing results because polymorphism of the copies may wrongly lead to the conclusion that different types are present in a specimen. To determine the copy number per genome of the nuclear rDNA and beta-tubulin genes analyzed for the typing of Pneumocystis carinii f. sp. hominis, we developed a strategy based on the use of the same multicompetitor molecule in two different quantitative-competitive PCRs, one for the gene under study and the other for a reference single copy gene, allowing direct comparison of the results of both PCRs. Control experiments showed that the strategy was sensitive enough to detect duplication of a gene. The copy number of the nuclear rDNA operon was determined by amplification of the intron of the 26S rDNA gene and that of the beta-tubulin by amplification of the region surrounding the intron no. 6. The method was first tested on P. c. carinii, the special form commonly infecting rats. Pneumocystis c. carinii was found to contain a single copy of the rDNA operon. The method was then applied to P. c. hominis. The results confirmed that P. c. hominis genome contains a single copy of the nuclear rDNA and beta-tubulin genes.