Development of a gene-based radiation hybrid map of chicken Chromosome 7 and comparison to human and mouse

To validate the ChickRH6 whole-genome radiation hybrid (WGRH) panel, we constructed a map of chicken Chromosome 7 based on 19 microsatellite markers from the genetic map and 76 ESTs (expressed sequence tags), whose efficient targeted development was made possible by using the ICCARE software. This high-density radiation hybrid (RH) map of a chicken macrochromosome gives us indications on characteristics of ChickRH6. The potential resolution of the panel is 325 kb and the practical resolution of our framework map is 1.3 Mb. Based on these results, a complete framework map of the chicken genome would comprise 1000 markers. The marker order is in good agreement with the genetic map and comparison with the human and mouse sequence maps revealed a number of internal rearrangements.     

Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes

We studied in rats the expression of genes involved in gluconeogenesis from glutamine and glycerol in the small intestine (SI) during fasting and diabetes. From Northern blot and enzymatic studies, we report that only phosphoenolpyruvate carboxykinase (PEPCK) activity is induced at 24 h of fasting, whereas glucose-6-phosphatase (G-6-Pase) activity is induced only from 48 h. Both genes then plateau, whereas glutaminase and glycerokinase strikingly rebound between 48 and 72 h. The two latter genes are fully expressed in streptozotocin-diabetic rats. From arteriovenous balance and isotopic techniques, we show that the SI does not release glucose at 24 h of fasting and that SI gluconeogenesis contributes to 35% of total glucose production in 72-h-fasted rats. The new findings are that 1) the SI can quantitatively account for up to one-third of glucose production in prolonged fasting; 2) the induction of PEPCK is not sufficient by itself to trigger SI gluconeogenesis; 3) G-6-Pase likely plays a crucial role in this process; and 4) glutaminase and glycerokinase may play a key potentiating role in the latest times of fasting and in diabetes.     

Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis

Radiation enteritis, a common complication of radiation therapy for abdominal and pelvic cancers, is characterized by severe transmural fibrosis associated with mesenchymal cell activation, tissue disorganization, and deposition of fibrillar collagen. To investigate the mechanisms involved in this pathological accumulation of extracellular matrix, we studied gene expression of matrix components along with that of genes involved in matrix remodeling, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Hybrid selection on high-density cDNA array, real-time RT-PCR, gelatin zymography and immunohistochemistry were used to characterize the mRNA expression profile, activity, and tissue location of extracellular matrix-related genes in radiation enteritis compared with healthy ileum. cDNA array analysis revealed a strong induction of genes coding for collagens I, III, IV, VI, and VIII, SPARC, and tenascin-C, extracellular-matrix degrading enzymes (MMP-1, -2, -3, -14, -18+19), and metalloproteinase inhibitors (TIMP-1, -2, plasminogen activator inhibitor-1) in radiation enteritis. This increase was correlated with the degree of infiltration of the mucosa by inflammatory cells, and the presence of differentiated mesenchymal cells in the submucosa and muscularis propria. Despite the fact that expression of collagens, MMPs, and TIMPs simultaneously increase, quantification of net collagen deposition shows an overall accumulation of collagen. Our results indicate that late radiation enteritis tissues are subjected to active process of fibrogenesis as well as fibrolysis, with a balance toward fibrogenesis. This demonstrates that established fibrotic tissue is not scarred fixed tissue but is subjected to a dynamic remodeling process.     

Activation of stress-responsive promoters by ionizing radiation for deployment in targeted gene therapy

Radiotherapy is one of the principal modalities of cancer treatment, but the delivery of a curative dose of ionizing radiation (IR) to the tumour is frequently limited by the need to protect the normal tissues within the irradiated area from radiation damage. This problem could be circumvented if tumour cells could be selectively sensitized to killing by IR. One way to achieve this goal would be to transduce the tumour cells with expression vectors carrying toxin genes under the control of promoters that are inactive unless induced by IR. For this approach to be successful, two parameters must be met: (i) the expression vector has to be delivered to the tumour or its immediate vicinity (e.g. its vasculature) and (ii) the promoter driving the expression of the toxin gene has to have negligible basal activity, yet has to be activated by clinically-achievable doses of IR. Several vectors that fulfil these criteria are currently reaching clinical trials. In this review, we examine the response of mammalian cells to IR, and the current status of radiation-induced suicide gene therapy that is dependent on this response.     

Nucleotide diversity of the <Emphasis Type="Italic">ZmPox3</Emphasis> maize peroxidase gene: Relationships between a MITE insertion in exon 2 and variation in forage maize digestibility

Background  

Polymorphisms were investigated within the ZmPox3 maize peroxidase gene, possibly involved in lignin biosynthesis because of its colocalization with a cluster of QTL related to lignin content and cell wall digestibility. The purpose of this study was to identify, on the basis of 37 maize lines chosen for their varying degrees of cell wall digestibility and representative of temperate regions germplasm, ZmPox3 haplotypes or individual polymorphisms possibly associated with digestibility.     

Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.     

HLM1, an essential signaling component in the hypersensitive response,is a member of the cyclic nucleotide-gated channel ion channel family

The hypersensitive response (HR) in plants is a programmed cell death that is commonly associated with disease resistance. A novel mutation in Arabidopsis, hlm1, which causes aberrant regulation of cell death, manifested by a lesion-mimic phenotype and an altered HR, segregated as a single recessive allele. Broad-spectrum defense mechanisms remained functional or were constitutive in the mutant plants, which also exhibited increased resistance to a virulent strain of Pseudomonas syringae pv tomato. In response to avirulent strains of the same pathogen, the hlm1 mutant showed differential abilities to restrict bacterial growth, depending on the avirulence gene expressed by the pathogen. The HLM1 gene encodes a cyclic nucleotide-gated channel, CNGC4. Preliminary study of the HLM1/CNGC4 gene pro-duct in Xenopus oocytes (inside-out patch-clamp technique) showed that CNGC4 is permeable to both K(+) and Na(+) and is activated by both cGMP and cAMP. HLM1 gene expression is induced in response to pathogen infection and some pathogen-related signals. Thus, HLM1 might constitute a common downstream component of the signaling pathways leading to HR/resistance.     

RLIP,an effector of the Ral GTPases,is a platform for Cdk1 to phosphorylate epsin during the switch off of endocytosis in mitosis

The Ral signaling pathway is critically involved in Ras-dependent oncogenesis. One of its key actors, RLIP/RalBP1, which participates in receptor endocytosis during interphase, is also involved in mitotic processes when endocytosis is switched off. During mitosis, RLIP76 is located on the duplicated centrosomes and is required for their proper separation and movement to the poles. We have looked for actors that associate with RLIP during mitosis. We show here that RLIP/RalBP1 interacts with an active p34cdc2.cyclinB1 (cdk1) enzyme and that this interaction is crucial for the mitotic phosphorylation of Epsin that, once phosphorylated, is no longer competent for endocytosis. We show also that this latter phosphorylation is dependent on Ral signaling. We propose that RLIP/RalBP1 is used as a platform by the mitotic cdk1 to facilitate the phosphorylation of Epsin, which makes Epsin incompetent for endocytosis during mitosis, when endocytosis is switched off.     

In vivo calpain/caspase cross-talk during 3-nitropropionic acid-induced striatal degeneration: implication of a calpain-mediated cleavage of active caspase-3

The role of caspases and calpains in neurodegeneration remains unclear. In this study, we focused on these proteases in a rat model of Huntington's disease using the mitochondrial toxin 3-nitropropionic acid (3NP). Results showed that 3NP-induced death of striatal neurons was preceded by cytochrome c redistribution, transient caspase-9 processing, and activation of calpain, whereas levels of the active/processed form of caspase-3 remained low and were even reduced as compared with control animals. We evidenced here that this decrease in active caspase-3 levels could be attributed to calpain activation. Several observations supported this conclusion. 1) Pharmacological blockade of calpain in 3NP-treated rats increased the levels of endogenous processed caspase-9 and caspase-3. 2) Cell-free extracts prepared from the striatum of 3NP-treated rats degraded in vitro the p34 and p20 subunits of active recombinant caspase-9 and caspase-3, respectively. 3) This degradation of p34 and p20 could be mimicked by purified mu-calpain and was prevented by calpain inhibitors. 4) mu-Calpain produced a loss of the DEVDase (Asp-Glu-Val-Asp) activity of active caspase-3. 5) Western blot analysis and experiments with 35S-radiolabeled caspase-3 showed that mu-calpain cleaved the p20 subunit of active caspase-3 near its catalytic site. 6) mu-Calpain activity was selectively inhibited (IC50 of 100 mum) by a 12 amino acid peptide corresponding to the C terminus of p20. Our results showed that calpain can down-regulate the caspase-9/caspase-3 cell death pathway during neurodegeneration due to chronic mitochondrial defects in vivo and that this effect may involve, at least in part, direct cleavage of the caspase-3 p20 subunit.